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- All Subjects: Bioengineering
- Creators: Stabenfeldt, Sarah
Description
The research question explored in this thesis is how CRISPR mediated editing is influenced by artificially opened chromatin in cells. Closed chromatin poses a barrier to Cas9 binding and editing at target genes. Synthetic pioneer factors (PFs) are a promising new approach to artificially open condensed heterochromatin allowing greater access of target DNA to Cas9. The Haynes lab has constructed fusions of enzymatic chromatin-modifying domains designed to remodel chromatin and increase Cas9 editing efficiency. With a library of PFs available, this research focuses on analyzing the behavior of Cas9 in chromatin that has been artificially opened by PFs. The types and frequency of INDELs (insertions & deletions) were determined after non-homologous end joining (NHEJ) in PF and Cas9-treated cells using quantitative Sanger sequencing and Synthego’s ICE software. Furthermore, NOME-seq analysis was carried out to map nucleosome position in PF and Cas9 treated cells. Although this experiment was unsuccessful, the heat map generated with data obtained from Synthego ICE predicts a possible presence of nucleosome in the vicinity suggesting that perhaps a fully open chromatin state was not achieved. Linear Regression analysis with certain assumptions confirms that with the increase in distance downstream of cut-site, the editing frequency decreases exponentially. Nevertheless, further experimental work should be carried out to investigate this hypothesis.
ContributorsHamna, Syeda Fatima (Author) / Haynes, Karmella A (Thesis advisor) / Stabenfeldt, Sarah (Thesis advisor) / Tian, Xiaojun (Committee member) / Arizona State University (Publisher)
Created2019
Description
Myocardial infarction (MI) remains the leading cause of mortality and morbidity in the U.S., accounting for nearly 140,000 deaths per year. Heart transplantation and implantation of mechanical assist devices are the options of last resort for intractable heart failure, but these are limited by lack of organ donors and potential surgical complications. In this regard, there is an urgent need for developing new effective therapeutic strategies to induce regeneration and restore the loss contractility of infarcted myocardium. Over the past decades, regenerative medicine has emerged as a promising strategy to develop scaffold-free cell therapies and scaffold-based cardiac patches as potential approaches for MI treatment. Despite the progress, there are still critical shortcomings associated with these approaches regarding low cell retention, lack of global cardiomyocytes (CMs) synchronicity, as well as poor maturation and engraftment of the transplanted cells within the native myocardium. The overarching objective of this dissertation was to develop two classes of nanoengineered cardiac patches and scaffold-free microtissues with superior electrical, structural, and biological characteristics to address the limitations of previously developed tissue models. An integrated strategy, based on micro- and nanoscale technologies, was utilized to fabricate the proposed tissue models using functionalized gold nanomaterials (GNMs). Furthermore, comprehensive mechanistic studies were carried out to assess the influence of conductive GNMs on the electrophysiology and maturity of the engineered cardiac tissues. Specifically, the role of mechanical stiffness and nano-scale topographies of the scaffold, due to the incorporation of GNMs, on cardiac cells phenotype, contractility, and excitability were dissected from the scaffold’s electrical conductivity. In addition, the influence of GNMs on conduction velocity of CMs was investigated in both coupled and uncoupled gap junctions using microelectrode array technology. Overall, the key contributions of this work were to generate new classes of electrically conductive cardiac patches and scaffold-free microtissues and to mechanistically investigate the influence of conductive GNMs on maturation and electrophysiology of the engineered tissues.
ContributorsNavaei, Ali (Author) / Nikkhah, Mehdi (Thesis advisor) / Brafman, David (Committee member) / Migrino, Raymond Q. (Committee member) / Stabenfeldt, Sarah (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2018
Description
Monitoring complex diseases and their comorbidities requires accurate and convenient measurements of multiple biomarkers. However, many state-of-the-art bioassays not only require complicated and time-consuming procedures, but also measure only one biomarker at a time. This noncomprehensive single-biomarker monitoring, as well as the cost and complexity of these bioassays advocate for a simple, rapid multi-marker sensing platform suitable for point-of-care or self-monitoring settings. To address this need, diabetes mellitus was selected as the example complex disease, with dry eye disease and cardiovascular disease as the example comorbidities. Seven vital biomarkers from these diseases were selected to investigate the platform technology: lactoferrin (Lfn), immunoglobulin E (IgE), insulin, glucose, lactate, low density lipoprotein (LDL), and high density lipoprotein (HDL). Using electrochemical techniques such as amperometry and electrochemical impedance spectroscopy (EIS), various single- and dual-marker sensing prototypes were studied. First, by focusing on the imaginary impedance of EIS, an analytical algorithm for the determination of optimal frequency and signal deconvolution was first developed. This algorithm helped overcome the challenge of signal overlapping in EIS multi-marker sensors, while providing a means to study the optimal frequency of a biomarker. The algorithm was then applied to develop various single- and dual-marker prototypes by exploring different kinds of molecular recognition elements (MRE) while studying the optimal frequencies of various biomarkers with respect to their biological properties. Throughout the exploration, 5 single-marker biosensors (glucose, lactate, insulin, IgE, and Lfn) and one dual-marker (LDL and HDL) biosensor were successfully developed. With the aid of nanoparticles and the engineering design of experiments, the zeta potential, conductivity, and molecular weight of a biomarker were found to be three example factors that contribute to a biomarker’s optimal frequency. The study platforms used in the study did not achieve dual-enzymatic marker biosensors (glucose and lactate) due to signal contamination from localized accumulation of reduced electron mediators on self-assembled monolayer. However, amperometric biosensors for glucose and lactate with disposable test strips and integrated samplers were successfully developed as a back-up solution to the multi-marker sensing platform. This work has resulted in twelve publications, five patents, and one submitted manuscripts at the time of submission.
ContributorsLin, Chi En (Author) / La Belle, Jeffrey T (Thesis advisor) / Caplan, Michael (Committee member) / Cook, Curtiss B (Committee member) / Stabenfeldt, Sarah (Committee member) / Spano, Mark (Committee member) / Arizona State University (Publisher)
Created2018
Description
One of the most prominent biological challenges for the field of drug delivery is the blood-brain barrier. This physiological system blocks the entry of or actively removes almost all small molecules into the central nervous system (CNS), including many drugs that could be used to treat diseases in the CNS. Previous studies have shown that activation of the adenosine receptor signaling pathway through the use of agonists has been demonstrated to increase BBB permeability. For example, regadenoson is an adenosine A2A receptor agonist that has been shown to disrupt the BBB and allow for increased drug uptake in the CNS. The goal of this study was to verify this property of regadenoson. We hypothesized that co-administration of regadenoson with a non-brain penetrant macromolecule would facilitate its entry into the central nervous system. To test this hypothesis, healthy mice were administered regadenoson or saline concomitantly with a fluorescent dextran solution. The brain tissue was either homogenized to measure quantity of fluorescent molecule, or cryosectioned for imaging with confocal fluorescence microscopy. These experiments did not identify any significant difference in the amount of fluorescence detected in the brain after regadenoson treatment. These results contradict those of previous studies and highlight potential differences in injection methodology, time windows, and properties of brain impermeant molecules.
ContributorsWohlleb, Gregory Michael (Author) / Sirianni, Rachael (Thesis director) / Stabenfeldt, Sarah (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2015-05
Description
Traumatic brain injury (TBI) is a leading cause of death in individuals under the age of 45, resulting in over 50,000 deaths each year. Over 80,000 TBI patients report long-term deficits consisting of motor or cognitive dysfunctions due to TBI pathophysiology. The biochemical secondary injury triggers a harmful inflammatory cascade, gliosis, and astrocyte activation surrounding the injury lesion, and no current treatments exist to alleviate these underlying pathologies. In order to mitigate the negative inflammatory effects of the secondary injury, we created a hydrogel comprised of hyaluronic acid (HA) and laminin, and we hypothesized that the anti-inflammatory properties of HA will decrease astrocyte activation and inflammation after TBI. C57/BL6 mice were subjected to mild-to-moderate CCI. Three days following injury, mice were treated with injection of vehicle or HA-Laminin hydrogel. Mice were sacrificed at three and seven days post injection and analyzed for astrocyte and inflammatory responses. In mice treated with vehicle injections, astrocyte activation was significantly increased at three days post-transplantation in the injured cortex and injury lesion. However, mice treated with the HA-Laminin hydrogel experienced significantly reduced acute astrocyte activation at the injury site three days post transplantation. Interestingly, there were no significant differences in astrocyte activation at seven days post treatment in either group. Although the microglial and macrophage response remains to be investigated, our data suggest that the HA-Laminin hydrogel demonstrates potential for TBI therapeutics targeting inflammation, including acute modulation of the astrocyte, microglia, and macrophage response to TBI.
ContributorsGoddery, Emma Nicole (Author) / Stabenfeldt, Sarah (Thesis director) / Addington, Caroline (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Neurological disorders are difficult to treat with current drug delivery methods due to their inefficiency and the lack of knowledge of the mechanisms behind drug delivery across the blood brain barrier (BBB). Nanoparticles (NPs) are a promising drug delivery method due to their biocompatibility and ability to be modified by cell penetrating peptides, such as transactivating transciptor (TAT) peptide, which has been shown to increase efficiency of delivery. There are multiple proposed mechanisms of TAT-mediated delivery that also have size restrictions on the molecules that can undergo each BBB crossing mechanism. The effect of nanoparticle size on TAT-mediated delivery in vivo is an important aspect to research in order to better understand the delivery mechanisms and to create more efficient NPs. NPs called FluoSpheres are used because they come in defined diameters unlike polymeric NPs that have a broad distribution of diameters. Both modified and unmodified 100nm and 200nm NPs were able to bypass the BBB and were seen in the brain, spinal cord, liver, and spleen using confocal microscopy and a biodistribution study. Statistically significant differences in delivery rate of the different sized NPs or between TAT-modified and unmodified NPs were not found. Therefore in future work a larger range of diameter size will be evaluated. Also the unmodified NPs will be conjugated with scrambled peptide to ensure that both unmodified and TAT-modified NPs are prepared in identical fashion to better understand the role of size on TAT targeting. Although all the NPs were able to bypass the BBB, future work will hopefully provide a better representation of how NP size effects the rate of TAT-mediated delivery to the CNS.
ContributorsCeton, Ricki Ronea (Author) / Stabenfeldt, Sarah (Thesis director) / Sirianni, Rachael (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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