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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
Few-layer black phosphorous (FLBP) is one of the most important two-dimensional (2D) materials due to its strongly layer-dependent quantized bandstructure, which leads to wavelength-tunable optical and electrical properties. This thesis focuses on the preparation of stable, high-quality FLBP, the characterization of its optical properties, and device applications.Part I presents an approach to preparing high-quality, stable FLBP samples by combining O2 plasma etching, boron nitride (BN) sandwiching, and subsequent rapid thermal annealing (RTA). Such a strategy has successfully produced FLBP samples with a record-long lifetime, with 80% of photoluminescence (PL) intensity remaining after 7 months. The improved material quality of FLBP allows the establishment of a more definitive relationship between the layer number and PL energies.
Part II presents the study of oxygen incorporation in FLBP. The natural oxidation formed in the air environment is dominated by the formation of interstitial oxygen and dangling oxygen. By the real-time PL and Raman spectroscopy, it is found that continuous laser excitation breaks the bonds of interstitial oxygen, and free oxygen atoms can diffuse around or form dangling oxygen under low heat. RTA at 450 °C can turn the interstitial oxygen into dangling oxygen more thoroughly. Such oxygen-containing samples show similar optical properties to the pristine BP samples. The bandgap of such FLBP samples increases with the concentration of the incorporated oxygen.
Part III deals with the investigation of emission natures of the prepared samples. The power- and temperature-dependent measurements demonstrate that PL emissions are dominated by excitons and trions, with a combined percentage larger than 80% at room temperature. Such measurements allow the determination of trion and exciton binding energies of 2-, 3-, and 4-layer BP, with values around 33, 23, 15 meV for trions and 297, 276, 179 meV for excitons at 77K, respectively.
Part IV presents the initial exploration of device applications of such FLBP samples. The coupling between photonic crystal cavity (PCC) modes and FLBP's emission is realized by integrating the prepared sandwich structure onto 2D PCC. Electroluminescence has also been achieved by integrating such materials onto interdigital electrodes driven by alternating electric fields.
ContributorsLi, Dongying (Author) / Ning, Cun-Zheng (Thesis advisor) / Vasileska, Dragica (Committee member) / Lai, Ying-Cheng (Committee member) / Yu, Hongbin (Committee member) / Arizona State University (Publisher)
Created2022
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
Predicting nonlinear dynamical systems has been a long-standing challenge in science. This field is currently witnessing a revolution with the advent of machine learning methods. Concurrently, the analysis of dynamics in various nonlinear complex systems continues to be crucial. Guided by these directions, I conduct the following studies. Predicting critical transitions and transient states in nonlinear dynamics is a complex problem. I developed a solution called parameter-aware reservoir computing, which uses machine learning to track how system dynamics change with a driving parameter. I show that the transition point can be accurately predicted while trained in a sustained functioning regime before the transition. Notably, it can also predict if the system will enter a transient state, the distribution of transient lifetimes, and their average before a final collapse, which are crucial for management. I introduce a machine-learning-based digital twin for monitoring and predicting the evolution of externally driven nonlinear dynamical systems, where reservoir computing is exploited. Extensive tests on various models, encompassing optics, ecology, and climate, verify the approach’s effectiveness. The digital twins can extrapolate unknown system dynamics, continually forecast and monitor under non-stationary external driving, infer hidden variables, adapt to different driving waveforms, and extrapolate bifurcation behaviors across varying system sizes. Integrating engineered gene circuits into host cells poses a significant challenge in synthetic biology due to circuit-host interactions, such as growth feedback. I conducted systematic studies on hundreds of circuit structures exhibiting various functionalities, and identified a comprehensive categorization of growth-induced failures. I discerned three dynamical mechanisms behind these circuit failures. Moreover, my comprehensive computations reveal a scaling law between the circuit robustness and the intensity of growth feedback. A class of circuits with optimal robustness is also identified. Chimera states, a phenomenon of symmetry-breaking in oscillator networks, traditionally have transient lifetimes that grow exponentially with system size. However, my research on high-dimensional oscillators leads to the discovery of ’short-lived’ chimera states. Their lifetime increases logarithmically with system size and decreases logarithmically with random perturbations, indicating a unique fragility. To understand these states, I use a transverse stability analysis supported by simulations.
ContributorsKong, Lingwei (Author) / Lai, Ying-Cheng (Thesis advisor) / Tian, Xiaojun (Committee member) / Papandreou-Suppappola, Antonia (Committee member) / Alkhateeb, Ahmed (Committee member) / Arizona State University (Publisher)
Created2023
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
A notable challenge when assembling synthetic gene circuits is that modularity often fails to function as intended. A crucial underlying reason for this modularity failure is the existence of competition for shared and limited gene expression resources. By designing a synthetic cascading bistable switches (Syn-CBS) circuit in a single strain with two coupled self-activation modules to achieve successive cell fate transitions, nonlinear resource competition within synthetic gene circuits is unveiled. However, in vivo it can be seen that the transition path was redirected with the activation of one switch always prevailing over that of the other, contradictory to coactivation theoretically expected. This behavior is a result of resource competition between genes and follows a ‘winner-takes-all’ rule, where the winner is determined by the relative connection strength between the two modules. Despite investigation demonstrating that resource competition between gene modules can significantly alter circuit deterministic behaviors, how resource competition contributes to gene expression noise and how this noise can be controlled is still an open issue of fundamental importance in systems biology and biological physics. By utilizing a two-gene circuit, the effects of resource competition on protein expression noise levels can be closely studied. A surprising double-edged role is discovered: the competition for these resources decreases noise while the constraint on resource availability adds its own term of noise into the system, denoted “resource competitive” noise. Noise reduction effects are then studied using orthogonal resources. Results indicate that orthogonal resources are a good strategy for eliminating the contribution of resource competition to gene expression noise.
Noise propagation through a cascading circuit has been considered without resource competition. It has been noted that the noise from upstream genes can be transmitted downstream. However, resource competition’s effects on this cascading noise have yet to be studied. When studied, it is found that resource competition can induce stochastic state
switching and perturb noise propagation. Orthogonal resources can remove some of the resource competitive behavior and allow for a system with less noise.
ContributorsGoetz, Hanah Elizabeth (Author) / Tian, Xiaojun (Thesis advisor) / Wang, Xiao (Committee member) / Lai, Ying-Cheng (Committee member) / Arizona State University (Publisher)
Created2022
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
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