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Description
Gene manipulation techniques, such as RNA interference (RNAi), offer a powerful method for elucidating gene function and discovery of novel therapeutic targets in a high-throughput fashion. In addition, RNAi is rapidly being adopted for treatment of neurological disorders, such as Alzheimer's disease (AD), Parkinson's disease, etc. However, a major challenge

Gene manipulation techniques, such as RNA interference (RNAi), offer a powerful method for elucidating gene function and discovery of novel therapeutic targets in a high-throughput fashion. In addition, RNAi is rapidly being adopted for treatment of neurological disorders, such as Alzheimer's disease (AD), Parkinson's disease, etc. However, a major challenge in both of the aforementioned applications is the efficient delivery of siRNA molecules, plasmids or transcription factors to primary cells such as neurons. A majority of the current non-viral techniques, including chemical transfection, bulk electroporation and sonoporation fail to deliver with adequate efficiencies and the required spatial and temporal control. In this study, a novel optically transparent biochip is presented that can (a) transfect populations of primary and secondary cells in 2D culture (b) readily scale to realize high-throughput transfections using microscale electroporation and (c) transfect targeted cells in culture with spatial and temporal control. In this study, delivery of genetic payloads of different sizes and molecular characteristics, such as GFP plasmids and siRNA molecules, to precisely targeted locations in primary hippocampal and HeLa cell cultures is demonstrated. In addition to spatio-temporally controlled transfection, the biochip also allowed simultaneous assessment of a) electrical activity of neurons, b) specific proteins using fluorescent immunohistochemistry, and c) sub-cellular structures. Functional silencing of GAPDH in HeLa cells using siRNA demonstrated a 52% reduction in the GAPDH levels. In situ assessment of actin filaments post electroporation indicated a sustained disruption in actin filaments in electroporated cells for up to two hours. Assessment of neural spike activity pre- and post-electroporation indicated a varying response to electroporation. The microarray based nature of the biochip enables multiple independent experiments on the same culture, thereby decreasing culture-to-culture variability, increasing experimental throughput and allowing cell-cell interaction studies. Further development of this technology will provide a cost-effective platform for performing high-throughput genetic screens.
ContributorsPatel, Chetan (Author) / Muthuswamy, Jitendran (Thesis advisor) / Helms Tillery, Stephen (Committee member) / Jain, Tilak (Committee member) / Caplan, Michael (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2012
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Description
A cerebral aneurysm is an abnormal ballooning of the blood vessel wall in the brain that occurs in approximately 6% of the general population. When a cerebral aneurysm ruptures, the subsequent damage is lethal damage in nearly 50% of cases. Over the past decade, endovascular treatment has emerged as an

A cerebral aneurysm is an abnormal ballooning of the blood vessel wall in the brain that occurs in approximately 6% of the general population. When a cerebral aneurysm ruptures, the subsequent damage is lethal damage in nearly 50% of cases. Over the past decade, endovascular treatment has emerged as an effective treatment option for cerebral aneurysms that is far less invasive than conventional surgical options. Nonetheless, the rate of successful treatment is as low as 50% for certain types of aneurysms. Treatment success has been correlated with favorable post-treatment hemodynamics. However, current understanding of the effects of endovascular treatment parameters on post-treatment hemodynamics is limited. This limitation is due in part to current challenges in in vivo flow measurement techniques. Improved understanding of post-treatment hemodynamics can lead to more effective treatments. However, the effects of treatment on hemodynamics may be patient-specific and thus, accurate tools that can predict hemodynamics on a case by case basis are also required for improving outcomes.Accordingly, the main objectives of this work were 1) to develop computational tools for predicting post-treatment hemodynamics and 2) to build a foundation of understanding on the effects of controllable treatment parameters on cerebral aneurysm hemodynamics. Experimental flow measurement techniques, using particle image velocimetry, were first developed for acquiring flow data in cerebral aneurysm models treated with an endovascular device. The experimental data were then used to guide the development of novel computational tools, which consider the physical properties, design specifications, and deployment mechanics of endovascular devices to simulate post-treatment hemodynamics. The effects of different endovascular treatment parameters on cerebral aneurysm hemodynamics were then characterized under controlled conditions. Lastly, application of the computational tools for interventional planning was demonstrated through the evaluation of two patient cases.
ContributorsBabiker, M. Haithem (Author) / Frakes, David H (Thesis advisor) / Adrian, Ronald (Committee member) / Caplan, Michael (Committee member) / Chong, Brian (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2013
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Description
Coronary heart disease (CHD) is the most prevalent cause of death worldwide. Atherosclerosis which is the condition of plaque buildup on the inside of the coronary artery wall is the main cause of CHD. Rupture of unstable atherosclerotic coronary plaque is known to be the cause of acute coronary syndrome.

Coronary heart disease (CHD) is the most prevalent cause of death worldwide. Atherosclerosis which is the condition of plaque buildup on the inside of the coronary artery wall is the main cause of CHD. Rupture of unstable atherosclerotic coronary plaque is known to be the cause of acute coronary syndrome. The composition of plaque is important for detection of plaque vulnerability. Due to prognostic importance of early stage identification, non-invasive assessment of plaque characterization is necessary. Computed tomography (CT) has emerged as a non-invasive alternative to coronary angiography. Recently, dual energy CT (DECT) coronary angiography has been performed clinically. DECT scanners use two different X-ray energies in order to determine the energy dependency of tissue attenuation values for each voxel. They generate virtual monochromatic energy images, as well as material basis pair images. The characterization of plaque components by DECT is still an active research topic since overlap between the CT attenuations measured in plaque components and contrast material shows that the single mean density might not be an appropriate measure for characterization. This dissertation proposes feature extraction, feature selection and learning strategies for supervised characterization of coronary atherosclerotic plaques. In my first study, I proposed an approach for calcium quantification in contrast-enhanced examinations of the coronary arteries, potentially eliminating the need for an extra non-contrast X-ray acquisition. The ambiguity of separation of calcium from contrast material was solved by using virtual non-contrast images. Additional attenuation data provided by DECT provides valuable information for separation of lipid from fibrous plaque since the change of their attenuation as the energy level changes is different. My second study proposed these as the input to supervised learners for a more precise classification of lipid and fibrous plaques. My last study aimed at automatic segmentation of coronary arteries characterizing plaque components and lumen on contrast enhanced monochromatic X-ray images. This required extraction of features from regions of interests. This study proposed feature extraction strategies and selection of important ones. The results show that supervised learning on the proposed features provides promising results for automatic characterization of coronary atherosclerotic plaques by DECT.
ContributorsYamak, Didem (Author) / Akay, Metin (Thesis advisor) / Muthuswamy, Jit (Committee member) / Akay, Yasemin (Committee member) / Pavlicek, William (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2013
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Description
Gold nanoparticles have emerged as promising nanomaterials for biosensing, imaging, photothermal treatment and therapeutic delivery for several diseases, including cancer. We have generated poly(amino ether)-functionalized gold nanorods (PAE-GNRs) using a layer-by-layer deposition approach. Sub-toxic concentrations of PAE-GNRs were employed to deliver plasmid DNA to prostate cancer cells in vitro. PAE-GNRs

Gold nanoparticles have emerged as promising nanomaterials for biosensing, imaging, photothermal treatment and therapeutic delivery for several diseases, including cancer. We have generated poly(amino ether)-functionalized gold nanorods (PAE-GNRs) using a layer-by-layer deposition approach. Sub-toxic concentrations of PAE-GNRs were employed to deliver plasmid DNA to prostate cancer cells in vitro. PAE-GNRs generated using 1,4C-1,4Bis, a cationic polymer from our laboratory demonstrated significantly higher transgene expression and exhibited lower cytotoxicities when compared to similar assemblies generated using 25 kDa poly(ethylene imine) (PEI25k-GNRs), a current standard for polymer-mediated gene delivery. Additionally, sub-toxic concentrations of 1,4C-1,4Bis-GNR nanoassemblies were employed to deliver expression vectors that express shRNA ('shRNA plasmid') against firefly luciferase gene in order to knock down expression of the protein constitutively expressed in prostate cancer cells. The roles of poly(amino ether) chemistry and zeta-potential in determining transgene expression efficacies of PAE-GNR assemblies were investigated. The theranostic potential of 1,4C-1,4Bis-GNR nanoassemblies was demonstrated using live cell two-photon induced luminescence bioimaging. The PAE class of polymers was also investigated for the one pot synthesis of both gold and silver nanoparticles using a small library poly(amino ethers) derived from linear-like polyamines. Efficient nanoparticle synthesis dependent on concentration of polymers as well as polymer chemical composition is demonstrated. Additionally, the application of poly(amino ether)-gold nanoparticles for transgene delivery is demonstrated in 22Rv1 and MB49 cancer cell lines. Base polymer, 1,4C-1,4Bis and 1,4C-1,4Bis templated and modified gold nanoparticles were compared for transgene delivery efficacies. Differences in morphology and physiochemical properties were investigated as they relate to differences in transgene delivery efficacy. There were found to be minimal differences suggestion that 1,4C-1,4Bis efficacy is not lost following use for nanoparticle modification. These results indicate that poly(amino ether)-gold nanoassemblies are a promising theranostic platform for delivery of therapeutic payloads capable of simultaneous gene silencing and bioimaging.
ContributorsRamos, James (Author) / Rege, Kaushal (Thesis advisor) / Kodibagkar, Vikram (Committee member) / Caplan, Michael (Committee member) / Vernon, Brent (Committee member) / Garcia, Antonio (Committee member) / Arizona State University (Publisher)
Created2014
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Description
In this work, a novel method is developed for making nano- and micro- fibrous hydrogels capable of preventing the rejection of implanted materials. This is achieved by either (1) mimicking the native cellular environment, to exert fine control over the cellular response or (2) acting as a protective barrier, to

In this work, a novel method is developed for making nano- and micro- fibrous hydrogels capable of preventing the rejection of implanted materials. This is achieved by either (1) mimicking the native cellular environment, to exert fine control over the cellular response or (2) acting as a protective barrier, to camouflage the foreign nature of a material and evade recognition by the immune system. Comprehensive characterization and in vitro studies described here provide a foundation for developing substrates for use in clinical applications. Hydrogel dextran and poly(acrylic acid) (PAA) fibers are formed via electrospinning, in sizes ranging from nanometers to microns in diameter. While "as-electrospun" fibers are continuous in length, sonication is used to fragment fibers into short fiber "bristles" and generate nano- and micro- fibrous surface coatings over a wide range of topographies. Dex-PAA fibrous surfaces are chemically modified, and then optimized and characterized for non-fouling and ECM-mimetic properties. The non-fouling nature of fibers is verified, and cell culture studies show differential responses dependent upon chemical, topographical and mechanical properties. Dex-PAA fibers are advantageously unique in that (1) a fine degree of control is possible over three significant parameters critical for modifying cellular response: topography, chemistry and mechanical properties, over a range emulating that of native cellular environments, (2) the innate nature of the material is non-fouling, providing an inert background for adding back specific bioactive functionality, and (3) the fibers can be applied as a surface coating or comprise the scaffold itself. This is the first reported work of dex-PAA hydrogel fibers formed via electrospinning and thermal cross-linking, and unique to this method, no toxic solvents or cross-linking agents are needed to create hydrogels or for surface attachment. This is also the first reported work of using sonication to fragment electrospun hydrogel fibers, and in which surface coatings were made via simple electrostatic interaction and dehydration. These versatile features enable fibrous surface coatings to be applied to virtually any material. Results of this research broadly impact the design of biomaterials which contact cells in the body by directing the consequent cell-material interaction.
ContributorsLouie, Katherine BoYook (Author) / Massia, Stephen P (Thesis advisor) / Bennett, Kevin (Committee member) / Garcia, Antonio (Committee member) / Pauken, Christine (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2011
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Description
Aqueous solutions of temperature-responsive copolymers based on N-isopropylacrylamide (NIPAAm) hold promise for medical applications because they can be delivered as liquids and quickly form gels in the body without organic solvents or chemical reaction. However, their gelation is often followed by phase-separation and shrinking. Gel shrinking and water loss is

Aqueous solutions of temperature-responsive copolymers based on N-isopropylacrylamide (NIPAAm) hold promise for medical applications because they can be delivered as liquids and quickly form gels in the body without organic solvents or chemical reaction. However, their gelation is often followed by phase-separation and shrinking. Gel shrinking and water loss is a major limitation to using NIPAAm-based gels for nearly any biomedical application. In this work, a graft copolymer design was used to synthesize polymers which combine the convenient injectability of poly(NIPAAm) with gel water content controlled by hydrophilic side-chain grafts based on Jeffamine® M-1000 acrylamide (JAAm). The first segment of this work describes the synthesis and characterization of poly(NIPAAm-co-JAAm) copolymers which demonstrates controlled swelling that is nearly independent of LCST. The graft copolymer design was then used to produce a degradable antimicrobial-eluting gel for prevention of prosthetic joint infection. The resorbable graft copolymer gels were shown to have three unique characteristics which demonstrate their suitability for this application. First, antimicrobial release is sustained and complete within 1 week. Second, the gels behave like viscoelastic fluids, enabling complete surface coverage of an implant without disrupting fixation or movement. Finally, the gels degrade rapidly within 1-6 weeks, which may enable their use in interfaces where bone healing takes place. Graft copolymer hydrogels were also developed which undergo Michael addition in situ with poly(ethylene glycol) diacrylate to form elastic gels for endovascular embolization of saccular aneurysms. Inclusion of JAAm grafts led to weaker physical crosslinking and faster, more complete chemical crosslinking. JAAm grafts prolonged the delivery window of the system from 30 seconds to 220 seconds, provided improved gel swelling, and resulted in stronger, more elastic gels within 30 minutes after delivery.
ContributorsOverstreet, Derek (Author) / Caplan, Michael (Thesis advisor) / Massia, Stephen (Committee member) / Mclaren, Alexander (Committee member) / Vernon, Brent (Committee member) / McLemore, Ryan (Committee member) / Arizona State University (Publisher)
Created2012
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Description
Biosensors offer excellent diagnostic methods through precise quantification of bodily fluid biomarkers and could fill an important niche in diagnostic screening. The long term goal of this research is the development of an impedance immunosensor for easy-to-use, rapid, sensitive and selective simultaneously multiplexed quantification of bodily fluid disease biomarkers. To

Biosensors offer excellent diagnostic methods through precise quantification of bodily fluid biomarkers and could fill an important niche in diagnostic screening. The long term goal of this research is the development of an impedance immunosensor for easy-to-use, rapid, sensitive and selective simultaneously multiplexed quantification of bodily fluid disease biomarkers. To test the hypothesis that various cytokines induce empirically determinable response frequencies when captured by printed circuit board (PCB) impedance immunosensor surface, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) methods were used to test PCB biosensors versus multiple cytokine biomarkers to determine limits of detection, background interaction and response at all sweep frequencies. Results indicated that sensors for cytokine Interleukin-12 (IL-12) detected their target over three decades of concentration and were tolerant to high levels of background protein. Further, the hypothesis that cytokine analytes may be rapidly detected via constant frequency impedance immunosensing without sacrificing undue sensitivity, CV, EIS, impedance-time (Zt) methods and modeling were used to test CHITM gold electrodes versus IL-12 over different lengths of time to determine limits of detection, detection time, frequency of response and consistent cross-platform sensor performance. Modeling and Zt studies indicate interrogation of the electrode with optimum frequency could be used for detection of different target concentrations within 90 seconds of sensor exposure and that interrogating the immunosensor with fixed, optimum frequency could be used for sensing target antigen. This informs usability of fixed-frequency impedance methods for biosensor research and particularly for clinical biosensor use. Finally, a multiplexing impedance immunosensor prototype for quantification of biomarkers in various body fluids was designed for increased automation of sample handling and testing. This enables variability due to exogenous factors and increased rapidity of assay with eased sensor fabrication. Methods were provided for simultaneous multiplexing through multisine perturbation of a sensor, and subsequent data processing. This demonstrated ways to observe multiple types of antibody-antigen affinity binding events in real time, reducing the number of sensors and target sample used in the detection and quantification of multiple biomarkers. These features would also improve the suitability of the sensor for clinical multiplex detection of disease biomarkers.
ContributorsFairchild, Aaron (Author) / La Belle, Jeffrey T (Thesis advisor) / Muthuswamy, Jitendran (Committee member) / Nagaraj, Vinay (Committee member) / Pizziconi, Vince (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2012
Description
Cooperativity can be used to manipulate binding affinities of DNA biosensors – improving specificity without sacrificing sensitivity; examples include tentacle probes (TPs) and cooperative primers (CPs). This thesis body of work: (1) used TPs to develop a rapid, low-cost diagnostic for detecting the point mutation leading to Navajo Neurohepatopathy (NNH)

Cooperativity can be used to manipulate binding affinities of DNA biosensors – improving specificity without sacrificing sensitivity; examples include tentacle probes (TPs) and cooperative primers (CPs). This thesis body of work: (1) used TPs to develop a rapid, low-cost diagnostic for detecting the point mutation leading to Navajo Neurohepatopathy (NNH) and (2) used CPs to amplify a symmetric bowtie-barcoded origami with captured t-cell receptor (TCR) α and β mRNA of a single cell.

NNH (affecting 1-in-1600 Navajo babies) is a fatal genetic disorder often caused by 149G>A mutation and is characterized by brain damage and liver disease/failure. Phoenix Children’s Hospital currently uses gene sequencing to identify the 149G>A mutation. While this process is conclusive, there are limitations, as it requires both time (3-4 weeks) and money (>$700). Ultimately, these factors create barriers that can directly impact a patient’s quality of life. Assessment of the developed TP diagnostic, using genomic DNA derived from FFPE patient liver samples, suggests nearly 100% specificity and sensitivity while reducing cost to ~$250 (including cost of labor) and providing a diagnosis within 48 hours.

TCR specificity is dependent on V(D)J recombination as well as pairing of the αβ chains. Drs. Schoettle and Blattman have developed a solution in which a bowtie-barcoded origami strand nanostructure is transfected into individual cells of a heterogeneous cell population to capture and protect αβ mRNA. When PCR of the origami template is performed with Vα, X, Vβ, and Y primers, the α and β gene segments cannot be tied back to a barcode – and paired. Assessment of the developed CPs for PCR suggests correct individual amplification using (1) Va + Xcp and (2) Vβ + Ycp primers, whereas combination of all the primers (Va, Xcp, Vb, and Ycp) suggests hybridization of the Vα + Xcp and Vβ + Ycp products due to the origami target symmetry.
ContributorsDubois, Courtney Michelle (Author) / Caplan, Michael R (Thesis advisor) / Vernon, Brent (Committee member) / Carpentieri, David (Committee member) / Arizona State University (Publisher)
Created2018
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Description
Minimally invasive endovascular embolization procedures decrease surgery time, speed up recovery, and provide the possibility for more comprehensive treatment of aneurysms, arteriovenous malformations (AVMs), and hypervascular tumors. Liquid embolic agents (LEAs) are preferred over mechanical embolic agents, such as coils, because they achieve homogeneous filling of aneurysms and more complex

Minimally invasive endovascular embolization procedures decrease surgery time, speed up recovery, and provide the possibility for more comprehensive treatment of aneurysms, arteriovenous malformations (AVMs), and hypervascular tumors. Liquid embolic agents (LEAs) are preferred over mechanical embolic agents, such as coils, because they achieve homogeneous filling of aneurysms and more complex angioarchitectures. The gold standard of commercially available LEAs is dissolved in dimethyl sulfoxide (DMSO), which has been associated with vasospasm and angiotoxicity. The aim of this study was to investigate amino acid substitution in an enzyme-degradable side group of an N-isopropylacrylamide (NIPAAm) copolymer for the development of a LEA that would be delivered in water and degrade at the rate that tissue is regenerated. NIPAAm copolymers have a lower critical solution temperature (LCST) due to their amphiphilic nature. This property enables them to be delivered as liquids through a microcatheter below their LCST and to solidify in situ above the LCST, which would result in the successful selective occlusion of blood vessels. Therefore, in this work, a series of poly(NIPAAm-co-peptide) copolymers with hydrophobic side groups containing the Ala-Pro-Gly-Leu collagenase substrate peptide sequence were synthesized as in situ forming, injectable copolymers.. The Gly-Leu peptide bond in these polypeptides is cleaved by collagenase, converting the side group into the more hydrophilic Gly-Ala-Pro-Gly-COOH (GAPG-COOH), thus increasing the LCST of the hydrogel after enzyme degradation. Enzyme degradation property and moderate mechanical stability convinces the use of these copolymers as liquid embolic agents.
ContributorsRosas Gomez, Karime Jocelyn (Author) / Vernon, Brent (Thesis advisor) / Weaver, Jessica (Committee member) / Pal, Amrita (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

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