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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 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
Description
The purpose of this study was to determine the feasibility of a mindfulness-based intervention among pregnant women (12-20 weeks’ gestation) using a mobile meditation app, Calm. This study involved 100 participants who were recruited nationally due to the COVID-19 pandemic. This study was reviewed and approved by the Institutional Review Board of Arizona State University (STUDY STUDY00010467). All participants were provided an informed consent document and provided electronic consent prior to enrollment and participation in this study. This study was a randomized, controlled trial (trial registration: ClinicalTrials.gov NCT04264910). Participants randomized to the intervention group were asked to participate in a minimum of 10 minutes of daily meditation using a mindfulness meditation mobile app (i.e., Calm) for the duration of their pregnancy. Participants randomized to the standard of care control group were given access to the app after they gave birth. Both the intervention and control groups were administered surveys that measured feasibility outcomes, perceived stress, mindfulness, self-compassion, impact from COVID-19, pregnancy-related anxiety, depression, emotional regulation, sleep, and childbirth experience at four time points: baseline (12-20 weeks gestation), midline (24 weeks gestation), postintervention (36 weeks gestation), and follow-up survey (3-5 weeks postpartum). Data is currently being analyzed for publication.
ContributorsLister, Haily (Author) / Huberty, Jennifer (Thesis director) / Larkey, Linda (Committee member) / School of Mathematical and Natural Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
APOE encodes for a lipid transport protein and has three allelic variants-APOE ε2, ε3 and ε4 each of which differentially modulate the risk for Alzheimer’s disease (AD). The presence of the ε4 allele of APOE greatly increases AD risk compared to the presence of the more prevalent and risk neutral ε3 allele. An imbalance in the generation and clearance of amyloid beta (Aβ) peptides has been hypothesized to play a key role in driving the disease. APOE4 impacts several AD-relevant cellular processes. However, it is unclear whether these effects represent a gain of toxic function or a loss of function, specifically as it relates to modulating amyloid beta (Aβ) levels. Here, a set of APOE knockout (KO) and APOE4 isogenic human induced pluripotent stem cells (hiPSCs) were generated from a parental APOE3 hiPSC line with a highly penetrant familial AD (fAD) mutation to investigate this with respect to Aβ secretion in neural cultures and Aβ uptake in monocultures of microglia-like cells (iMGLs). Conversion of APOE3 to E4 as well as functionally knocking APOE out from the APOE3 parental line, result in elevated Aβ levels in neural cultures, likely through multiple mechanisms including the altered processing of the precursor protein to Aβ called amyloid precursor protein (APP). In pure neuronal cultures, a shift in the processing of APP was observed with the Aβ-generating amyloidogenic pathway being favored in both APOE3 as well as APOE4 neurons compared to APOE KO neurons, with APOE4 neurons exhibiting a greater shift. In iMGLs derived from the isogenic hiPSC lines, expression of APOE, regardless of the isoform, lowered the uptake of Aβ. Overall, APOE4 modulates Aβ levels through distinct loss of protective and gain of function effects. Dissecting these effects would contribute towards a better understanding of the design of potential APOE-targeted therapeutics in the future.
ContributorsRajaram Srinivasan, Gayathri (Author) / Brafman, David (Thesis advisor) / Plaisier, Christopher (Committee member) / Newbern, Jason (Committee member) / Stabenfeldt, Sarah (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2024
Description
College students experience a considerable amount of stress. Unmanaged stress is associated with poor academic performance, health risk behaviors (i.e., inadequate sleep and physical activity, alcohol consumption, poor dietary behaviors), and poor mental health. Coping with stress has become a priority among universities. The most tested stress-related programs to date have been mindfulness-based and face-to-face. These programs demonstrated significant improvements in stress, mindfulness, and self-compassion among college students. However, they may be burdensome to students as studies report low attendance and low compliance due to class conflicts or not enough time. Few interventions have used more advanced technologies (i.e., mobile apps) as a mode of delivery. The purpose of this study is to report adherence to a consumer-based mindfulness meditation mobile application (i.e., Calm) and test its effects on stress, mindfulness, and self-compassion in college students. We will also explore what the relationship is between mindfulness and health behaviors.
College students were recruited using fliers on college campus and social media. Eligible participants were randomized to one of two groups: (1) Intervention - meditate using Calm, 10 min/day for eight weeks and (2) Control – no participation in mindfulness practices (received the Calm application after 12-weeks). Stress, mindfulness, and self-compassion and health behaviors (i.e., sleep disturbance, alcohol consumption, physical activity, fruit and vegetable consumption) were measured using self-report. Outcomes were measured at baseline and week eight.
Of the 109 students that enrolled in the study, 41 intervention and 47 control participants were included in analysis. Weekly meditation participation averaged 38 minutes with 54% of participants completing at least half (i.e., 30 minutes) of meditations. Significant changes between groups were found in stress, mindfulness, and self-compassion (all P<0.001) in favor of the intervention group. A significant negative association (p<.001) was found between total mindfulness and sleep disturbance.
An eight-week consumer-based mindfulness meditation mobile application (i.e., Calm) was effective in reducing stress, improving mindfulness and self-compassion among undergraduate college students. Mobile applications may be a feasible, effective, and less burdensome way to reduce stress in college students.
College students were recruited using fliers on college campus and social media. Eligible participants were randomized to one of two groups: (1) Intervention - meditate using Calm, 10 min/day for eight weeks and (2) Control – no participation in mindfulness practices (received the Calm application after 12-weeks). Stress, mindfulness, and self-compassion and health behaviors (i.e., sleep disturbance, alcohol consumption, physical activity, fruit and vegetable consumption) were measured using self-report. Outcomes were measured at baseline and week eight.
Of the 109 students that enrolled in the study, 41 intervention and 47 control participants were included in analysis. Weekly meditation participation averaged 38 minutes with 54% of participants completing at least half (i.e., 30 minutes) of meditations. Significant changes between groups were found in stress, mindfulness, and self-compassion (all P<0.001) in favor of the intervention group. A significant negative association (p<.001) was found between total mindfulness and sleep disturbance.
An eight-week consumer-based mindfulness meditation mobile application (i.e., Calm) was effective in reducing stress, improving mindfulness and self-compassion among undergraduate college students. Mobile applications may be a feasible, effective, and less burdensome way to reduce stress in college students.
ContributorsGlissmann, Christine (Author) / Huberty, Jennifer (Thesis advisor) / Sebren, Ann (Committee member) / Larkey, Linda (Committee member) / Lee, Chong (Committee member) / Arizona State University (Publisher)
Created2018
Description
Synthetic manipulation of chromatin dynamics has applications for medicine, agriculture, and biotechnology. However, progress in this area requires the identification of design rules for engineering chromatin systems. In this thesis, I discuss research that has elucidated the intrinsic properties of histone binding proteins (HBP), and apply this knowledge to engineer novel chromatin binding effectors. Results from the experiments described herein demonstrate that the histone binding domain from chromobox protein homolog 8 (CBX8) is portable and can be customized to alter its endogenous function. First, I developed an assay to identify engineered fusion proteins that bind histone post translational modifications (PTMs) in vitro and regulate genes near the same histone PTMs in living cells. This assay will be useful for assaying the function of synthetic histone PTM-binding actuators and probes. Next, I investigated the activity of a novel, dual histone PTM binding domain regulator called Pc2TF. I characterized Pc2TF in vitro and in cells and show it has enhanced binding and transcriptional activation compared to a single binding domain fusion called Polycomb Transcription Factor (PcTF). These results indicate that valency can be used to tune the activity of synthetic histone-binding transcriptional regulators. Then, I report the delivery of PcTF fused to a cell penetrating peptide (CPP) TAT, called CP-PcTF. I treated 2D U-2 OS bone cancer cells with CP-PcTF, followed by RNA sequencing to identify genes regulated by CP-PcTF. I also showed that 3D spheroids treated with CP-PcTF show delayed growth. This preliminary work demonstrated that an epigenetic effector fused to a CPP can enable entry and regulation of genes in U-2 OS cells through DNA independent interactions. Finally, I described and validated a new screening method that combines the versatility of in vitro transcription and translation (IVTT) expressed protein coupled with the histone tail microarrays. Using Pc2TF as an example, I demonstrated that this assay is capable of determining binding and specificity of a synthetic HBP. I conclude by outlining future work toward engineering HBPs using techniques such as directed evolution and rational design. In conclusion, this work outlines a foundation to engineer and deliver synthetic chromatin effectors.
ContributorsTekel, Stefan (Author) / Haynes, Karmella (Thesis advisor) / Mills, Jeremy (Committee member) / Caplan, Michael (Committee member) / Brafman, David (Committee member) / Arizona State University (Publisher)
Created2019
Description
Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, or amyotrophic lateral sclerosis are defined by the loss of several types of neurons and glial cells within the central nervous system (CNS). Combatting these diseases requires a robust population of relevant cell types that can be employed in cell therapies, drug screening, or patient specific disease modeling. Human induced pluripotent stem cells (hiPSC)-derived neural progenitor cells (hNPCs) have the ability to self-renew indefinitely and differentiate into the various neuronal and glial cell types of the CNS. In order to realize the potential of hNPCs, it is necessary to develop a xeno-free scalable platform for effective expansion and differentiation. Previous work in the Brafman lab led to the engineering of a chemically defined substrate—vitronectin derived peptide (VDP), which allows for the long-term expansion and differentiation of hNPCs. In this work, we use this substrate as the basis for a microcarrier (MC)-based suspension culture system. Several independently derived hNPC lines were cultured on MCs for multiple passages as well as efficiently differentiated to neurons. Finally, this MC-based system was used in conjunction with a low shear rotating wall vessel (RWV) bioreactor for the integrated, large-scale expansion and neuronal differentiation of hNPCs. Finally, VDP was shown to support the differentiation of hNPCs into functional astrocytes. Overall, this fully defined and scalable biomanufacturing system will facilitate the generation of hNPCs and their derivatives in quantities necessary for basic and translational applications.
ContributorsMorgan, Daylin (Author) / Brafman, David (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2018
Description
Calcium imaging is a well-established, non-invasive or minimally technique designed to study the electrical signaling neurons. Calcium regulates the release of gliotransmitters in astrocytes. Analyzing astrocytic calcium transients can provide significant insights into mechanisms such as neuroplasticity and neural signal modulation.
In the past decade, numerous methods have been developed to analyze in-vivo calcium imaging data that involves complex techniques such as overlapping signals segregation and motion artifact correction. The hypothesis used to detect calcium signal is the spatiotemporal sparsity of calcium signal, and these methods are unable to identify the passive cells that are not actively firing during the time frame in the video. Statistics regarding the percentage of cells in each frame of view can be critical for the analysis of calcium imaging data for human induced pluripotent stem cells derived neurons and astrocytes.
The objective of this research is to develop a simple and efficient semi-automated pipeline for analysis of in-vitro calcium imaging data. The region of interest (ROI) based image segmentation is used to extract the data regarding intensity fluctuation caused by calcium concentration changes in each cell. It is achieved by using two approaches: basic image segmentation approach and a machine learning approach. The intensity data is evaluated using a custom-made MATLAB that generates statistical information and graphical representation of the number of spiking cells in each field of view, the number of spikes per cell and spike height.
In the past decade, numerous methods have been developed to analyze in-vivo calcium imaging data that involves complex techniques such as overlapping signals segregation and motion artifact correction. The hypothesis used to detect calcium signal is the spatiotemporal sparsity of calcium signal, and these methods are unable to identify the passive cells that are not actively firing during the time frame in the video. Statistics regarding the percentage of cells in each frame of view can be critical for the analysis of calcium imaging data for human induced pluripotent stem cells derived neurons and astrocytes.
The objective of this research is to develop a simple and efficient semi-automated pipeline for analysis of in-vitro calcium imaging data. The region of interest (ROI) based image segmentation is used to extract the data regarding intensity fluctuation caused by calcium concentration changes in each cell. It is achieved by using two approaches: basic image segmentation approach and a machine learning approach. The intensity data is evaluated using a custom-made MATLAB that generates statistical information and graphical representation of the number of spiking cells in each field of view, the number of spikes per cell and spike height.
ContributorsBhandarkar, Siddhi Umesh (Author) / Brafman, David (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Tian, Xiaojun (Committee member) / Arizona State University (Publisher)
Created2019
Description
Background: Although childhood engagement in physical activity has received growing attention, most children still do not meet the recommended daily 60 minutes of moderate to vigorous physical activity [MVPA]. Children of ethnic minorities are less likely to meet the guidelines. Interventions have been implemented in various settings to increase child physical activity levels, yet these efforts have not yielded consistent results. The purpose of this study was to assess the preliminary effects of a community-based intervention on light physical activity and MVPA among 6-11 year old children. Methods: The present study was part of a larger study called Athletes for Life [AFL], a family-based, nutrition-education and physical activity intervention. The present study focused on physical activity data from the first completed cohort of participants (n=29). This study was a randomized control trial in which participating children were randomized into a control (n=14) or intervention (n=15) group. Participants wore accelerometers at two time points. Intervention strategies were incorporated to increase child habitual physical activity. Analyses of covariance were performed to test for post 12-week differences between both groups on the average minutes of light physical activity and MVPA minutes per day.
Results: The accelerometer data demonstrated no significant difference in light physical activity or MVPA mean minutes per day between the groups. Few children reported engaging in activities sufficient for meeting the physical activity guidelines outside the AFL program. Of the 119 total distributed child physical activity tracker sheets (7 per family), 55 were returned. Of the 55 returned physical activity tracker sheets, parents reported engaging in physical activity with their children only 7 times outside of the program over seven weeks.
Conclusion: The combined intervention strategies implemented throughout the 12-week study did not appear to be effective at increasing habitual mean minutes per day spent engaging in light and MVPA among children beyond the directed program. Methodological limitations and low adherence to intervention strategies may partially explain these findings. Further research is needed to test successful strategies within community programs to increase habitual light physical activity and MVPA among 6-11 year old children.
Results: The accelerometer data demonstrated no significant difference in light physical activity or MVPA mean minutes per day between the groups. Few children reported engaging in activities sufficient for meeting the physical activity guidelines outside the AFL program. Of the 119 total distributed child physical activity tracker sheets (7 per family), 55 were returned. Of the 55 returned physical activity tracker sheets, parents reported engaging in physical activity with their children only 7 times outside of the program over seven weeks.
Conclusion: The combined intervention strategies implemented throughout the 12-week study did not appear to be effective at increasing habitual mean minutes per day spent engaging in light and MVPA among children beyond the directed program. Methodological limitations and low adherence to intervention strategies may partially explain these findings. Further research is needed to test successful strategies within community programs to increase habitual light physical activity and MVPA among 6-11 year old children.
ContributorsQuezada, Blanca (Author) / Crespo, Noe (Thesis advisor) / Huberty, Jennifer (Committee member) / Vega-Lopez, Sonia (Committee member) / Arizona State University (Publisher)
Created2015
Description
Several debilitating neurological disorders, such as Alzheimer's disease, stroke, and spinal cord injury, are characterized by the damage or loss of neuronal cell types in the central nervous system (CNS). Human neural progenitor cells (hNPCs) derived from human pluripotent stem cells (hPSCs) can proliferate extensively and differentiate into the various neuronal subtypes and supporting cells that comprise the CNS. As such, hNPCs have tremendous potential for disease modeling, drug screening, and regenerative medicine applications. However, the use hNPCs for the study and treatment of neurological diseases requires the development of defined, robust, and scalable methods for their expansion and neuronal differentiation. To that end a rational design process was used to develop a vitronectin-derived peptide (VDP)-based substrate to support the growth and neuronal differentiation of hNPCs in conventional two-dimensional (2-D) culture and large-scale microcarrier (MC)-based suspension culture. Compared to hNPCs cultured on ECMP-based substrates, hNPCs grown on VDP-coated surfaces displayed similar morphologies, growth rates, and high expression levels of hNPC multipotency markers. Furthermore, VDP surfaces supported the directed differentiation of hNPCs to neurons at similar levels to cells differentiated on ECMP substrates. Here it has been demonstrated that VDP is a robust growth and differentiation matrix, as demonstrated by its ability to support the expansions and neuronal differentiation of hNPCs derived from three hESC (H9, HUES9, and HSF4) and one hiPSC (RiPSC) cell lines. Finally, it has been shown that VDP allows for the expansion or neuronal differentiation of hNPCs to quantities (>1010) necessary for drug screening or regenerative medicine purposes. In the future, the use of VDP as a defined culture substrate will significantly advance the clinical application of hNPCs and their derivatives as it will enable the large-scale expansion and neuronal differentiation of hNPCs in quantities necessary for disease modeling, drug screening, and regenerative medicine applications.
ContributorsVarun, Divya (Author) / Brafman, David (Thesis advisor) / Nikkhah, Mehdi (Committee member) / Stabenfeldt, Sarah (Committee member) / Arizona State University (Publisher)
Created2016