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Most daily living tasks consist of pairing a series of sequential movements, e.g., reaching to a cup, grabbing the cup, lifting and returning the cup to your mouth. The process by which we control and mediate the smooth progression of these tasks is not well understood. One method which we can use to further evaluate these motions is known as Startle Evoked Movements (SEM). SEM is an established technique to probe the motor learning and planning processes by detecting muscle activation of the sternocleidomastoid muscles of the neck prior to 120ms after a startling stimulus is presented. If activation of these muscles was detected following a stimulus in the 120ms window, the movement is classified as Startle+ whereas if no sternocleidomastoid activation is detected after a stimulus in the allotted time the movement is considered Startle-. For a movement to be considered SEM, the activation of movements for Startle+ trials must be faster than the activation of Startle- trials. The objective of this study was to evaluate the effect that expertise has on sequential movements as well as determining if startle can distinguish when the consolidation of actions, known as chunking, has occurred. We hypothesized that SEM could distinguish words that were solidified or chunked. Specifically, SEM would be present when expert typists were asked to type a common word but not during uncommon letter combinations. The results from this study indicated that the only word that was susceptible to SEM, where Startle+ trials were initiated faster than Startle-, was an uncommon task "HET" while the common words "AND" and "THE" were not. Additionally, the evaluation of the differences between each keystroke for common and uncommon words showed that Startle was unable to distinguish differences in motor chunking between Startle+ and Startle- trials. Explanations into why these results were observed could be related to hand dominance in expert typists. No proper research has been conducted to evaluate the susceptibility of the non-dominant hand's fingers to SEM, and the results of future studies into this as well as the results from this study can impact our understanding of sequential movements.
ContributorsMieth, Justin Richard (Author) / Honeycutt, Claire (Thesis director) / Santello, Marco (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Alzheimer’s Disease (AD) affects over 5 million individuals in the U.S. and has a direct cost estimated in excess of $200 billion per year. Broadly speaking, there are two forms of AD—early-onset, familial AD (FAD) and late-onset-sporadic AD (SAD). Animal models of AD, which rely on the overexpression of FAD-related mutations, have provided important insights into the disease. However, these models do not display important disease-related pathologies and have been limited in their ability to model the complex genetics associated with SAD.
Advances in cellular reprogramming, have enabled the generation of in vitro disease models that can be used to dissect disease mechanisms and evaluate potential therapeutics. To that end, efforts by many groups, including the Brafman laboratory, to generated patient-specific hiPSCs have demonstrated the promise of studying AD in a simplified and accessible system. However, neurons generated from these hiPSCs have shown some, but not all, of the early molecular and cellular hallmarks associated with the disease. Additionally, phenotypes and pathological hallmarks associated with later stages of the human disease have not been observed with current hiPSC-based systems. Further, disease relevant phenotypes in neurons generated from SAD hiPSCs have been highly variable or largely absent. Finally, the reprogramming process erases phenotypes associated with cellular aging and, as a result, iPSC-derived neurons more closely resemble fetal brain rather than adult brain.
It is well-established that in vivo cells reside within a complex 3-D microenvironment that plays a significant role in regulating cell behavior. Signaling and other cellular functions, such as gene expression and differentiation potential, differ in 3-D cultures compared with 2-D substrates. Nonetheless, previous studies using AD hiPSCs have relied on 2-D neuronal culture models that do not reflect the 3-D complexity of native brain tissue, and therefore, are unable to replicate all aspects of AD pathogenesis. Further, the reprogramming process erases cellular aging phenotypes. To address these limitations, this project aimed to develop bioengineering methods for the generation of 3-D organoid-based cultures that mimic in vivo cortical tissue, and to generate an inducible gene repression system to recapitulate cellular aging hallmarks.
Advances in cellular reprogramming, have enabled the generation of in vitro disease models that can be used to dissect disease mechanisms and evaluate potential therapeutics. To that end, efforts by many groups, including the Brafman laboratory, to generated patient-specific hiPSCs have demonstrated the promise of studying AD in a simplified and accessible system. However, neurons generated from these hiPSCs have shown some, but not all, of the early molecular and cellular hallmarks associated with the disease. Additionally, phenotypes and pathological hallmarks associated with later stages of the human disease have not been observed with current hiPSC-based systems. Further, disease relevant phenotypes in neurons generated from SAD hiPSCs have been highly variable or largely absent. Finally, the reprogramming process erases phenotypes associated with cellular aging and, as a result, iPSC-derived neurons more closely resemble fetal brain rather than adult brain.
It is well-established that in vivo cells reside within a complex 3-D microenvironment that plays a significant role in regulating cell behavior. Signaling and other cellular functions, such as gene expression and differentiation potential, differ in 3-D cultures compared with 2-D substrates. Nonetheless, previous studies using AD hiPSCs have relied on 2-D neuronal culture models that do not reflect the 3-D complexity of native brain tissue, and therefore, are unable to replicate all aspects of AD pathogenesis. Further, the reprogramming process erases cellular aging phenotypes. To address these limitations, this project aimed to develop bioengineering methods for the generation of 3-D organoid-based cultures that mimic in vivo cortical tissue, and to generate an inducible gene repression system to recapitulate cellular aging hallmarks.
ContributorsBounds, Lexi Rose (Author) / Brafman, David (Thesis director) / Wang, Xiao (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Genetic manipulation of human cell lines has widespread applications in biomedical research ranging from disease modeling to therapeutic development. Human cells are generally difficult to genetically engineer, but exogenous nucleic acids can be expressed by viral, chemical, or nonchemical means. Chemical transfections are simpler in practice than both viral and nonchemical delivery of genetic material, but often suffer from cytotoxicity and low efficiency. Novel aminoglycoside antibiotic-derived lipopolymers have been synthesized to mediate transgene delivery to human cells. These polymers are comprised of either paromomycin or apramycin crosslinked with glycerol diglycidylether and derivatized with stearoyl chloride in varying molar ratios. In this work, three previously identified target lipopolymers were screened against a library of human embryonic and induced pluripotent stem cell lines. Cells were transfected with a plasmid encoding green fluorescent protein (GFP) and expression was quantified with flow cytometry 48 hours after transfection. Transfection efficiency was evaluated between three distinct lipopolymers and four lipopolymer:DNA mass ratios. GFP expression was compared to that of cells transfected with commercially available chemical gene delivery reagent controls\u2014JetPEI, Lipofectamine, and Fugene\u2014at their recommended reagent:DNA ratios. Improved transgene expression in stem cell lines allows for improved research methods. Human stem cell-derived neurons that have been genetically manipulated to express phenotypic characteristics of aging can be utilized to model neurodegenerative diseases, elucidating information about these diseases that would be inaccessible in unmanipulated tissue.
ContributorsMehta, Frea (Author) / Brafman, David (Thesis director) / Rege, Kaushal (Committee member) / Chemical Engineering Program (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Startle-evoked-movement (SEM), the involuntary release of a planned movement via a startling stimulus, has gained significant attention recently for its ability to probe motor planning as well as enhance movement of the upper extremity following stroke. We recently showed that hand movements are susceptible to SEM. Interestingly, only coordinated movements of the hand (grasp) but not individuated movements of the finger (finger abduction) were susceptible. It was suggested that this resulted from different neural mechanisms involved in each task; however it is possible this was the result of task familiarity. The objective of this study was to evaluate a more familiar individuated finger movement, typing, to determine if this task was susceptible to SEM. We hypothesized that typing movements will be susceptible to SEM in all fingers. These results indicate that individuated movements of the fingers are susceptible to SEM when the task involves a more familiar task, since the electromyogram (EMG) latency is faster in SCM+ trials compared to SCM- trials. However, the middle finger does not show a difference in terms of the keystroke voltage signal, suggesting the middle finger is less susceptible to SEM. Given that SEM is thought to be mediated by the brainstem, specifically the reticulospinal tract, this suggest that the brainstem may play a role in movements of the distal limb when those movements are very familiar, and the independence of each finger might also have a significant on the effect of SEM. Further research includes understanding SEM in fingers in the stroke population. The implications of this research can impact the way upper extremity rehabilitation is delivered.
ContributorsQuezada Valladares, Maria Jose (Author) / Honeycutt, Claire (Thesis director) / Santello, Marco (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
the project led by Professor Emma Frow, researching of stem cell clinics focused on stem cell applications, adherence to FDA guidelines, and characterization of information available and physician credentials. Regenerative medicine clinics commonly offered stem cell therapy, but introduced platelet rich plasma (PRP) and prolotherapy as regenerative therapies.
PRP and Prolotherapy are individual treatments that were even suggested and used in combination with stem cell therapies. Prolotherapy predates PRP as a chemical irritant therapy originally used to sclerose tissues. Prolotherapy is meant to stimulate platelet derived growth factors release to improve tissue healing response. Prolotherapy shows negligible efficacy improvements over corticosteroids, but may have underlying side effects from being an irritant. PRP is a more modern therapy for improved healing. Speculations state initial use was in an open heart surgery to improve healing post-surgery. PRP is created via centrifugation of patient blood to isolate growth factors by removing serum and other biological components to increase platelet concentration. PRP is comparable to corticosteroid injections in efficacy, but as an autologous application, there are no side effects making it more advantageous. Growth factors induce healing response and reduce inflammation. Growth factors stimulate cell growth, proliferation, differentiation, and stimulate cellular response mechanism such as angiogenesis and mitogenesis. The growth factor stimulation of PRP and prolotherapy both assist stem cell proliferation. Additional research is needed to determine differential capacity to ensure multipotent stem cells regenerate the correct cell type from the increased differential capacity offered by growth factor recruitment. The application of combination therapy for stem cells is unsubstantiated and applications violate FDA ‘minimal manipulation’ guidelines.
PRP and Prolotherapy are individual treatments that were even suggested and used in combination with stem cell therapies. Prolotherapy predates PRP as a chemical irritant therapy originally used to sclerose tissues. Prolotherapy is meant to stimulate platelet derived growth factors release to improve tissue healing response. Prolotherapy shows negligible efficacy improvements over corticosteroids, but may have underlying side effects from being an irritant. PRP is a more modern therapy for improved healing. Speculations state initial use was in an open heart surgery to improve healing post-surgery. PRP is created via centrifugation of patient blood to isolate growth factors by removing serum and other biological components to increase platelet concentration. PRP is comparable to corticosteroid injections in efficacy, but as an autologous application, there are no side effects making it more advantageous. Growth factors induce healing response and reduce inflammation. Growth factors stimulate cell growth, proliferation, differentiation, and stimulate cellular response mechanism such as angiogenesis and mitogenesis. The growth factor stimulation of PRP and prolotherapy both assist stem cell proliferation. Additional research is needed to determine differential capacity to ensure multipotent stem cells regenerate the correct cell type from the increased differential capacity offered by growth factor recruitment. The application of combination therapy for stem cells is unsubstantiated and applications violate FDA ‘minimal manipulation’ guidelines.
ContributorsKrum, Logan (Author) / Frow, Emma (Thesis director) / Brafman, David (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Research on human grasp typically involves the grasp of objects designed for the study of fingertip forces. Instrumented objects for such studies have often been designed for the simulation of functional tasks, such as feeding oneself, or for rigidity such that the objects do not deform when grasped. The goal of this thesis was to design a collapsible, instrumented object to study grasp of breakable objects. Such an object would enable experiments on human grip responses to unexpected finger-object events as well as anticipatory mechanisms once object fragility has been observed. The collapsible object was designed to be modular to allow for properties such as friction and breaking force to be altered. The instrumented object could be used to study both human and artificial grasp.
ContributorsTorrez, Troy (Author) / Santos, Veronica (Thesis director) / Santello, Marco (Committee member) / Artemiadis, Panagiotis (Committee member) / Barrett, The Honors College (Contributor)
Created2012-05
Description
Current research into live-cell dynamics, particularly those relating to chromatin structure and remodeling, are limited. The tools that are used to detect state changes in chromatin, such as Chromatin Immunoprecipitation and qPCR, require that the cell be killed off. This limits the ability of researchers to pinpoint changes in live cells over a longer period of time. As such, there is a need for a live-cell sensor that can detect chromatin state changes. The Chromometer is a transgenic chromatin state sensor designed to better understand human cell fate and the chromatin changes that occur. HOXD11.12, a DNA sequence that attracts repressive Polycomb group (PCG) proteins, was placed upstream of a core promoter-driven fluorescent reporter (AmCyan fluorescent protein, CFP) to link chromatin repression to a CFP signal. The transgene was stably inserted at an ectopic site in U2-OS (osteosarcoma) cells. Expression of CFP should reflect the epigenetic state at the HOXD locus, where several genes are regulated by Polycomb to control cell differentiation. U2-OS cells were transfected with the transgene and grown under selective pressure. Twelve colonies were identified as having integrated parts from the transgene into their genomes. PCR testing verified 2 cell lines that contain the complete transgene. Flow cytometry indicated mono-modal and bimodal populations in all transgenic cell colonies. Further research must be done to determine the effectiveness of this device as a sensor for live cell state change detection.
ContributorsBarclay, David (Co-author) / Simper, Jan (Co-author) / Haynes, Karmella (Thesis director) / Brafman, David (Committee member) / School of Life Sciences (Contributor) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
The purpose of this paper was to systematically review current literature regarding the effect of hand splints on aesthetic outcomes for individuals with acquired hand deformities. Hand splints vary in form and function, and are used to maintain or ameliorate hand function and aesthetics. A literature search was performed on peer-reviewed publications that used splinting as an intervention for conservative hand improvement. Evidence from ten randomized clinical trials (published from 2003 to 2015) was evaluated for aesthetic improvement among a total of 659 subjects. Cosmetic outcomes were analyzed by a change in angle measurements, such as extensor lag, ulnar deviation, and passive and active range of motion. Of these ten studies, five focused on hand deformities caused by neurological impairment, while the other five measured those with musculoskeletal complications. Only two of the ten studies concluded that splinting could aesthetically improve the hands, and only one of these reporting statistical significance in its data. The data was not only limited in quantity, but was presented in heterogeneous formats. There was an extensive variation in measured outcomes, intervention protocols, follow-up times, and many other aspects of the studies; this dissimilarity led to difficulty in performing a systematic assessment. The majority of evidence concludes that splinting does not improve the appearance of deformities, however none directly investigated this measure. Therefore, further RCTs that include measurements of cosmetic traits are necessary to better quantify the effect of splinting for management of hand deformities. This review was the first of its kind to evaluate the correction of hand deformities using splints as an intervention.
ContributorsVale, Nicholas Marshall (Author) / Santello, Marco (Thesis director) / Skiba, Jeffry (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor, Contributor) / School of Biological and Health Systems Engineering (Contributor)
Created2017-05
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