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- All Subjects: Biomedical Engineering
- All Subjects: Biology
- Creators: Brafman, David
- Member of: ASU Electronic Theses and Dissertations
Description
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
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
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
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
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
Description
The portability of genetic tools from one organism to another is a cornerstone of synthetic biology. The shared biological language of DNA-to-RNA-to-protein allows for expression of polypeptide chains in phylogenetically distant organisms with little modification. The tools and contexts are diverse, ranging from catalytic RNAs in cell-free systems to bacterial proteins expressed in human cell lines, yet they exhibit an organizing principle: that genes and proteins may be treated as modular units that can be moved from their native organism to a novel one. However, protein behavior is always unpredictable; drop-in functionality is not guaranteed.
My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli.
1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin.
2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists.
My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli.
1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin.
2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists.
ContributorsDaer, René (Author) / Haynes, Karmella (Thesis advisor) / Brafman, David (Committee member) / Nielsen, David (Committee member) / Kiani, Samira (Committee member) / Arizona State University (Publisher)
Created2017
Description
Synthetic gene networks have evolved from simple proof-of-concept circuits to
complex therapy-oriented networks over the past fifteen years. This advancement has
greatly facilitated expansion of the emerging field of synthetic biology. Multistability is a
mechanism that cells use to achieve a discrete number of mutually exclusive states in
response to environmental inputs. However, complex contextual connections of gene
regulatory networks in natural settings often impede the experimental establishment of
the function and dynamics of each specific gene network.
In this work, diverse synthetic gene networks are rationally designed and
constructed using well-characterized biological components to approach the cell fate
determination and state transition dynamics in multistable systems. Results show that
unimodality and bimodality and trimodality can be achieved through manipulation of the
signal and promoter crosstalk in quorum-sensing systems, which enables bacterial cells to
communicate with each other.
Moreover, a synthetic quadrastable circuit is also built and experimentally
demonstrated to have four stable steady states. Experiments, guided by mathematical
modeling predictions, reveal that sequential inductions generate distinct cell fates by
changing the landscape in sequence and hence navigating cells to different final states.
Circuit function depends on the specific protein expression levels in the circuit.
We then establish a protein expression predictor taking into account adjacent
transcriptional regions’ features through construction of ~120 synthetic gene circuits
(operons) in Escherichia coli. The predictor’s utility is further demonstrated in evaluating genes’ relative expression levels in construction of logic gates and tuning gene expressions and nonlinear dynamics of bistable gene networks.
These combined results illustrate applications of synthetic gene networks to
understand the cell fate determination and state transition dynamics in multistable
systems. A protein-expression predictor is also developed to evaluate and tune circuit
dynamics.
complex therapy-oriented networks over the past fifteen years. This advancement has
greatly facilitated expansion of the emerging field of synthetic biology. Multistability is a
mechanism that cells use to achieve a discrete number of mutually exclusive states in
response to environmental inputs. However, complex contextual connections of gene
regulatory networks in natural settings often impede the experimental establishment of
the function and dynamics of each specific gene network.
In this work, diverse synthetic gene networks are rationally designed and
constructed using well-characterized biological components to approach the cell fate
determination and state transition dynamics in multistable systems. Results show that
unimodality and bimodality and trimodality can be achieved through manipulation of the
signal and promoter crosstalk in quorum-sensing systems, which enables bacterial cells to
communicate with each other.
Moreover, a synthetic quadrastable circuit is also built and experimentally
demonstrated to have four stable steady states. Experiments, guided by mathematical
modeling predictions, reveal that sequential inductions generate distinct cell fates by
changing the landscape in sequence and hence navigating cells to different final states.
Circuit function depends on the specific protein expression levels in the circuit.
We then establish a protein expression predictor taking into account adjacent
transcriptional regions’ features through construction of ~120 synthetic gene circuits
(operons) in Escherichia coli. The predictor’s utility is further demonstrated in evaluating genes’ relative expression levels in construction of logic gates and tuning gene expressions and nonlinear dynamics of bistable gene networks.
These combined results illustrate applications of synthetic gene networks to
understand the cell fate determination and state transition dynamics in multistable
systems. A protein-expression predictor is also developed to evaluate and tune circuit
dynamics.
ContributorsWu, Fuqing (Author) / Wang, Xiao (Thesis advisor) / Haynes, Karmella (Committee member) / Marshall, Pamela (Committee member) / Nielsen, David (Committee member) / Brafman, David (Committee member) / Arizona State University (Publisher)
Created2017
Description
The pathophysiology of Alzheimer’s disease (AD) remains difficult to precisely ascertain in part because animal models fail to fully recapitulate many aspects of the disease and postmortem studies do not allow for the study of the pathophysiology. In vitro models of AD generated with patient derived human induced pluripotent stem cells (hiPSCs) could provide new insight into disease mechanisms. Although many protocols exist to differentiate hiPSCs to neurons, standard practice relies on two-dimensional (2-D) systems, which do not accurately mimic the complexity and architecture of the in vivo brain microenvironment. This research aims to create three-dimensional (3-D) models of AD using hiPSCs, which would enhance the understanding of AD pathophysiology thereby, enabling the generation of effective therapeutics.
ContributorsLundeen, Rachel (Author) / Brafman, David (Thesis advisor) / Kiani, Samira (Committee member) / Ebrahimkhani, Mohammad (Committee member) / Arizona State University (Publisher)
Created2017
Description
An in vitro model of Alzheimer’s disease (AD) is required to study the poorly understood molecular mechanisms involved in the familial and sporadic forms of the disease. Animal models have previously proven to be useful in studying familial Alzheimer’s disease (AD) by the introduction of AD related mutations in the animal genome and by the overexpression of AD related proteins. The genetics of sporadic Alzheimer’s is however too complex to model in an animal model. More recently, AD human induced pluripotent stem cells (hiPSCs) have been used to study the disease in a dish. However, AD hiPSC derived neurons do not faithfully reflect all the molecular characteristics and phenotypes observed in the aged cells with neurodegenerative disease. The truncated form of nuclear protein Lamin-A, progerin, has been implicated in premature aging and is found in increasing concentrations as normal cells age. We hypothesized that by overexpressing progerin, we can cause cells to ‘age’ and display the neurodegenerative effects observed with aging in both diseased and normal cells. To answer this hypothesis, we first generated a retrovirus that allows for the overexpression of progerin in AD and non-demented control (NDC) hiPSC derived neural progenitor cells(NPCs). Subsequently, we generated a pure population of hNPCs that overexpress progerin and wild type lamin. Finally, we analyzed the presence of various age related phenotypes such as abnormal nuclear structure and the loss of nuclear lamina associated proteins to characterize ‘aging’ in these cells.
ContributorsRaman, Sreedevi (Author) / Brafman, David (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2017