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Cardiovascular disease (CVD) remains the leading cause of mortality, resulting in 1 out of 4 deaths in the United States at the alarming rate of 1 death every 36 seconds, despite great efforts in ongoing research. In vitro research to study CVDs has had limited success, due to lack of biomimicry and structural complexity of 2D models. As such, there is a critical need to develop a 3D, biomimetic human cardiac tissue within precisely engineered in vitro platforms. This PhD dissertation involved development of an innovative anisotropic 3D human stem cell-derived cardiac tissue on-a-chip model (i.e., heart on-a-chip), with an enhanced maturation tissue state, as demonstrated through extensive biological assessments. To demonstrate the potential of the platform to study cardiac-specific diseases, the developed heart on-a-chip was used to model myocardial infarction (MI) due to exposure to hypoxia. The successful induction of MI on-a-chip (heart attack-on-a-chip) was evidenced through fibrotic tissue response, contractile dysregulation, and transcriptomic regulation of key pathways.This dissertation also described incorporation of CRISPR/Cas9 gene-editing to create a human induced pluripotent stem cell line (hiPSC) with a mutation in KCNH2, the gene implicated in Long QT Syndrome Type 2 (LQTS2). This novel stem cell line, combined with the developed heart on-a-chip technology, led to creation of a 3D human cardiac on-chip tissue model of LQTS2 disease.. Extensive mechanistic biological and electrophysiological characterizations were performed to elucidate the mechanism of R531W mutation in KCNH2, significantly adding to existing knowledge about LQTS2. In summary, this thesis described creation of a LQTS2 cardiac on-a-chip model, incorporated with gene-edited hiPSC-cardiomyocytes and hiPSC-cardiac fibroblasts, to study mechanisms of LQTS2.
Overall, this dissertation provides broad impact for fundamental studies toward cardiac biological studies as well as drug screening applications. Specifically, the developed heart on-a-chip from this dissertation provides a unique alternative platform to animal testing and 2D studies that recapitulates the human myocardium, with capabilities to model critical CVDs to study disease mechanisms, and/or ultimately lead to development of future therapeutic strategies.
ContributorsVeldhuizen, Jaimeson (Author) / Nikkhah, Mehdi (Thesis advisor) / Brafman, David (Committee member) / Ebrahimkhani, Mo (Committee member) / Migrino, Raymond Q (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2021
Description
Skin wounds can be caused by traumatic lacerations or incisions which disrupt the structural and functional integrity of the skin. Wound closure and primary intention treatment of the wound as soon as possible is crucial to avoid or minimize the risk of infection that can result in a compromised healing rate or advanced functional intricacy. The gold standard treatment for skin wound healing is suturing. Light-activated tissue sealing is an appealing alternative to sutures as it seals the wound edges minimizing the risk of infection and scarring, especially when utilized along with biodegradable polymeric biomaterials in the wound bed. Silk fibroins can be used as a biodegradable biomaterial that possesses properties supporting cell migration and proliferation in the tissue it interacts with. In addition, histamine treatment is shown to have extensive effects on cellular functions promoting wound healing. Here, the evaluation of Laser-activated Sealants (LASE) consisting of silk fibroin films induced with Indocyanine Green dye in a wound sealed with laser in the presence of Histamine receptor agonists H1R, H2R and H4R take place. The results were evaluated using Trans-epidermal Water Loss (TEWL), histological and analytical techniques where immune cell biomarkers Arginase-1, Ly6G, iNOS, Alpha-SMA, Proliferating Cell Nuclear Antigen (PCNA), and E-Cadherin were used to study the activity of specific cells such as macrophages, neutrophils, and myofibroblasts that aid in wound healing. PBS was used as a control for histamine receptor agonists. It was found that TEWL increased when treated with H1 receptor agonists while decreasing significantly in H2R and H4R-treated wounds. Arginase-1 activity improved, while it displayed an inverse relationship compared to iNOS. H4R agonist escalated Alpha-SMA cells, while others did not have any significant difference. Ly6G activity depleted in all histamine agonists significantly, while PCNA and E-Cadherin failed to show a positive or negative effect.
ContributorsPatel, Dirghau Manishbhai (Author) / Rege, Kaushal (Thesis advisor) / Massia, Stephen (Committee member) / Brafman, David (Committee member) / Arizona State University (Publisher)
Created2022
Description
Mycobacterial infections, as represented by leprosy and tuberculosis, have persisted as human pathogens for millennia. Their environmental counterparts, nontuberculous mycobacteria (NTM), are commodious infectious agents endowed with extensive innate and acquired antimicrobial resistance. The current drug development process selects for antibiotics with high specificity for definitive targets within bacterial metabolic and replication pathways. Because these compounds demonstrate limited efficacy against mycobacteria, novel antimycobacterial agents with unconventional mechanisms of action were identified. Two highly resistant NTMs, Mycobacterium abscessus (Mabs) a rapid-growing respiratory, skin, and soft tissue pathogen, and Mycobacterium ulcerans (MU), the causative agent of Buruli ulcer, were selected as targets. Compounds that indicated antimicrobial activity against other highly resistant pathogens were selected for initial screening. Antimicrobial peptides (AMPs) have demonstrated activity against a variety of bacterial pathogens, including mycobacterial species. Designed antimicrobial peptides (dAMPs), rationally-designed and synthetic contingents, combine iterative features of natural AMPs to achieve superior antimicrobial activity in resistant pathogens. Initial screening identified two dAMPs, RP554 and RP557, with bactericidal activity against Mabs. Clay-associated ions have previously demonstrated bactericidal activity against MU. Synthetic and customizable aluminosilicates have also demonstrated adsorption of bacterial cells and toxins. On this basis, two aluminosilicate materials, geopolymers (GP) and ion-exchange nanozeolites (IE-nZeos), were screened for antimicrobial activity against MU and its fast-growing relative, Mycobacterium marinum (Mmar). GPs demonstrated adsorption of MU cells and mycolactone, a secreted, lipophilic toxin, whereas Cu-nZeos and Ag-nZeos demonstrated antibacterial activity against MU and Mmar. Cumulatively, these results indicate that an integrative drug selection process may yield a new generation of antimycobacterial agents.
ContributorsDermody, Roslyn June (Author) / Haydel, Shelley E (Thesis advisor) / Bean, Heather (Committee member) / Nickerson, Cheryl (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2022
Description
High throughput transcriptome data analysis like Single-cell Ribonucleic Acid sequencing (scRNA-seq) and Circular Ribonucleic Acid (circRNA) data have made significant breakthroughs, especially in cancer genomics. Analysis of transcriptome time series data is core in identifying time point(s) where drastic changes in gene transcription are associated with homeostatic to non-homeostatic cellular transition (tipping points). In Chapter 2 of this dissertation, I present a novel cell-type specific and co-expression-based tipping point detection method to identify target gene (TG) versus transcription factor (TF) pairs whose differential co-expression across time points drive biological changes in different cell types and the time point when these changes are observed. This method was applied to scRNA-seq data sets from a SARS-CoV-2 study (18 time points), a human cerebellum development study (9 time points), and a lung injury study (18 time points). Similarly, leveraging transcriptome data across treatment time points, I developed methodologies to identify treatment-induced and cell-type specific differentially co-expressed pairs (DCEPs). In part one of Chapter 3, I presented a pipeline that used a series of statistical tests to detect DCEPs. This method was applied to scRNA-seq data of patients with non-small cell lung cancer (NSCLC) sequenced across cancer treatment times. However, this pipeline does not account for correlations among multiple single cells from the same sample and correlations among multiple samples from the same patient. In Part 2 of Chapter 3, I presented a solution to this problem using a mixed-effect model. In Chapter 4, I present a summary of my work that focused on the cross-species analysis of circRNA transcriptome time series data. I compared circRNA profiles in neonatal pig and mouse hearts, identified orthologous circRNAs, and discussed regulation mechanisms of cardiomyocyte proliferation and myocardial regeneration conserved between mouse and pig at different time points.
ContributorsNyarige, Verah Mocheche (Author) / Liu, Li (Thesis advisor) / Wang, Junwen (Thesis advisor) / Dinu, Valentin (Committee member) / Arizona State University (Publisher)
Created2022
Description
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
Beta-Amyloid(Aβ) plaques and tau protein tangles in the brain are now widely recognized as the defining hallmarks of Alzheimer’s disease (AD), followed by structural atrophy detectable on brain magnetic resonance imaging (MRI) scans. However, current methods to detect Aβ/tau pathology are either invasive (lumbar puncture) or quite costly and not widely available (positron emission tomography (PET)). And one of the particular neurodegenerative regions is the hippocampus to which the influence of Aβ/tau on has been one of the research projects focuses in the AD pathophysiological progress.
In this dissertation, I proposed three novel machine learning and statistical models to examine subtle aspects of the hippocampal morphometry from MRI that are associated with Aβ /tau burden in the brain, measured using PET images. The first model is a novel unsupervised feature reduction model to generate a low-dimensional representation of hippocampal morphometry for each individual subject, which has superior performance in predicting Aβ/tau burden in the brain. The second one is an efficient federated group lasso model to identify the hippocampal subregions where atrophy is strongly associated with abnormal Aβ/Tau. The last one is a federated model for imaging genetics, which can identify genetic and transcriptomic influences on hippocampal morphometry. Finally, I stated the results of these three models that have been published or submitted to peer-reviewed conferences and journals.
ContributorsWu, Jianfeng (Author) / Wang, Yalin (Thesis advisor) / Li, Baoxin (Committee member) / Liang, Jianming (Committee member) / Wang, Junwen (Committee member) / Wu, Teresa (Committee member) / Arizona State University (Publisher)
Created2022
Description
Mycobacterium tuberculosis (Mtb), the etiological agent of the tuberculosis disease, is estimated to infect one-fourth of the human population and is responsible for 1.5 million deaths annually. The increased emergence of bacterial resistance to clinical interventions highlights the lack in development of novel antimicrobial therapeutics. Prototypical bacterial two-component systems (TCS) allow for sensing of extracellular stimuli and relay thereof to create a transcriptional response. The prrAB TCS is essential for viability in Mtb, presenting itself as an attractive novel drug target. In Mtb, PrrAB is involved in the adaptation to the intra-macrophage environment and recent work implicates PrrAB in the dosR-dependent hypoxia adaptation. This work defines a direct molecular and regulatory connection between Mtb PrrAB and the dosR-dependent hypoxia response. Using electrophoretic mobility shift assays combined with surface plasmon resonance, the Mtb dosR gene is established as a specific target of PrrA, corroborated by fluorescence reporter assays demonstrating a regulatory relationship. Considering the scarce understanding of prrAB essentiality in nontuberculous mycobacteria and the presence of multiple prrAB orthologs in Mycobacterium smegmatis and Mycobacterium abscessus, CRISPR interference was utilized to evaluate the essentiality of PrrAB beyond Mtb. prrAB was found to be inessential for viability in M. smegmatis yet required for in vitro growth. Conversely, M. abscessus prrAB repression led to enhanced in vitro growth. Diarylthiazole-48 (DAT-48) displayed decreased selectivity against M. abscessus but demonstrated enhanced intrinsic activity upon prrAB repression in M. abscessus. Lastly, to aid in the rapid determination of mycobacterial drug susceptibility and the detection of mycobacterial heteroresistance, the large volume scattering imaging (LVSim) platform was adapted for mycobacteria. Using LVSim, Mtb drug susceptibility was detected phenotypically within 6 hours, and clinically relevant mycobacterial heteroresistance was detected phenotypically within 10 generations. The data generated in these studies provide insight into the essential role of PrrAB in Mtb and its involvement in the dosR-dependent hypoxia adaptation, advance the understanding of mycobacterial PrrAB essentiality and PrrAB-associated mycobacterial growth dependency. These studies further establish molecular and mechanistic connection between PrrAB and DAT-48 in Mtb and M. abscessus and develop a rapid phenotypic drug susceptibility testing platform for mycobacteria.
ContributorsHaller, Yannik Alex (Author) / Haydel, Shelley E (Thesis advisor) / Bean, Heather (Committee member) / Nickerson, Cheryl (Committee member) / Plaisier, Christopher (Committee member) / Acharya, Abhinav (Committee member) / Arizona State University (Publisher)
Created2023
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