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Title: THE RESPONSE OF SATELLITE GLIAL CELLS TO P2X7 RECEPTOR ACTIVATION.
Creator: Kursewicz, Christina D, Lambert,Stephen, University of Central Florida
Abstract / Description: Satellite glial cells (SGCs) surround the cell bodies of neurons of the peripheral nervous system, including those of the sensory ganglia. Their close apposition to the neuronal soma allows for bi-directional communication between neurons and SGCs, which are thought to regulate neuronal activity. After nerve injury, SGCs in the dorsal root ganglia contribute to neuropathic pain. Although the mechanisms are not fully understood, SGCs show increased coupling via gap junctions, and communicate...
Show moreSatellite glial cells (SGCs) surround the cell bodies of neurons of the peripheral nervous system, including those of the sensory ganglia. Their close apposition to the neuronal soma allows for bi-directional communication between neurons and SGCs, which are thought to regulate neuronal activity. After nerve injury, SGCs in the dorsal root ganglia contribute to neuropathic pain. Although the mechanisms are not fully understood, SGCs show increased coupling via gap junctions, and communicate with the neuron via bi-directional purinergic signaling after nerve injury. The increased coupling between SGCs and neurons may have implications for chronic pain following peripheral nerve injury. In vivo studies suggest that injury through the administration of capsaicin to the sensory nerve endings causes SGCs to be activated and proliferate. We have shown that capsaicin treatment in an in vitro co-culture of sensory neurons and SGCs increased the expression of the proliferation marker, Ki-67 in the glia. Here, we examine whether purinergic signaling plays a role in the promotion of SGC proliferation.
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Date Issued: 2017
Identifier: CFH2000172, ucf:45960
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH2000172

Title: Cell Printing: An Effective Advancement for the Creation of um Size Patterns for Integration into Microfluidic BioMEMs Devices.
Creator: Aubin, Megan, Hickman, James, Coffey, Kevin, Lambert, Stephen, University of Central Florida
Abstract / Description: The Body-on-a-Chip (BoaC) is a microfluidic BioMEMs project that aims to replicate major organs of the human body on a chip, providing an in vitro drug testing platform without the need to resort to animal model testing. Using a human model also provides significantly more accurate drug response data, and may even open the door to personalized drug treatments. Microelectrode arrays integrated with human neuronal or human cardiac cells that are positioned on the electrodes are essential...
Show moreThe Body-on-a-Chip (BoaC) is a microfluidic BioMEMs project that aims to replicate major organs of the human body on a chip, providing an in vitro drug testing platform without the need to resort to animal model testing. Using a human model also provides significantly more accurate drug response data, and may even open the door to personalized drug treatments. Microelectrode arrays integrated with human neuronal or human cardiac cells that are positioned on the electrodes are essential components for BoaC systems. Fabricating these substrates relies heavily on chemically patterned surfaces to control the orientation and growth of the cells. Currently, cells are plated by hand onto the surface of the chemically patterned microelectrode arrays. The cells that land on the cytophobic 2-[Methoxy(Polyethyleneoxy)6-9Propyl]trimethoxysilane (PEG) coating die and detach from the surface, while the cells that land on the cytophilic diethylenetriamine (DETA) coating survive and attach to the surface exhibiting normal physiology and function. The current technique wastes a significant amount of cells, some of which are extremely expensive, and is labor intensive. Cell printing, the process of dispensing cells through a 3D printer, makes it possible to pinpoint the placement of cells onto the microelectrodes, drastically reducing the number of cells utilized. Scaled-up manufacturing is also possible due to the automation capabilities provided by printing. In this work, the specific conditions for printing each cell type is unique, the printing of human motoneurons, human sensory neurons and human cardiac cells was investigated. The viability and functionality of the printed cells are demonstrated by phase images, immunostaining and electrical signal recordings. The superior resolution of cell printing was then taken one step further by successfully printing two different cell types in close proximity to encourage controlled innervation and communication.
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Date Issued: 2017
Identifier: CFE0007390, ucf:52074
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007390

Title: Unraveling the role of Phenylethanolamine N-methyltransferase (Pnmt+) cells in-vivo.
Creator: Manja, Sanjana, Ebert, Steven, Kim, Yoon-Seong, Lambert, Stephen, University of Central Florida
Abstract / Description: Phenylethanolamine N-methyltransferase (Pnmt) is the enzyme that N-methylates norepinephrine to produce the stress hormone/neurotransmitter, epinephrine, which is abundantly expressed in adrenal glands. Developmental studies have also identified Pnmt expression in the embryonic heart and several areas of the brain, including brainstem, cerebellum, and hypothalamus. Thus, we hypothesize that selective ablation of Pnmt+ cells will have detrimental effects on cardiovascular, neuromuscular, and...
Show morePhenylethanolamine N-methyltransferase (Pnmt) is the enzyme that N-methylates norepinephrine to produce the stress hormone/neurotransmitter, epinephrine, which is abundantly expressed in adrenal glands. Developmental studies have also identified Pnmt expression in the embryonic heart and several areas of the brain, including brainstem, cerebellum, and hypothalamus. Thus, we hypothesize that selective ablation of Pnmt+ cells will have detrimental effects on cardiovascular, neuromuscular, and metabolic processes. To uncover the importance of Pnmt+ cells in vivo, we generated a novel Diphtheria Toxin A (DTA) suicide model (Pnmt+/Cre; R26+/DTA) to selectively ablate Pnmt-expressing (Pnmt+) cells in mice. Appearing normal at birth, Pnmt-Cre/DTA mice began to develop apparent cardiovascular, neurological, and metabolic impairments soon thereafter. To measure cardiac function, we performed quantitative echocardiography, electrocardiography (ECG), and blood pressure measurements. Key findings from these assessments indicated decreased left-ventricular performance, slowed atrioventricular conduction, and increased pulse pressure in the Pnmt-Cre/DTA ablation mice. These mice also showed signs of motor control deficits as early as one month, which progressively worsened with age. To assess these effects, we performed standard motor tests including hind-limb clasping, grip strength, and rotarod balance tests. Moreover, we found that the Pnmt-Cre/DTA mice ceased to gain weight shortly after puberty. The motor and metabolic deficits apparent in these animals suggested potential neurological impairments, and we thus undertook immunohistochemical staining experiments to determine the localization of Pnmt+ cells in the brain. Staining revealed Pnmt expression in the Purkinje cells of the cerebellum (motor), paraventricular nucleus of the hypothalamus (metabolic), and surprisingly extensive staining in the cerebral cortex. These results demonstrate that Pnmt+ cell contributions in the brain are much more extensive than previously thought. Overall, this work opens new pathways that will have substantial impacts on our understanding of the roles Pnmt+ cells play in normal development and disorders affecting cardiovascular, motor, and metabolic functions.
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Date Issued: 2019
Identifier: CFE0007495, ucf:52649
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007495

Title: Recursive Behavior Recording: Complex Motor Stereotypies and Anatomical Behavior Descriptions.
Creator: Bobbitt, Nathaniel, Vasquez, Eleazar, Lambert, Stephen, Hughes, Charles, University of Central Florida
Abstract / Description: A novel anatomical behavioral descriptive taxonomy improves motion capture in complex motor stereotypies (CMS) by indexing precise time data without degradation in the complexity of whole body movement in CMS. The absence of etiological explanation of complex motor stereotypies warrants the aggregation of a core CMS dataset to compare regulation of repetitive behaviors in the time domain. A set of visual formalisms trap configurations of behavioral markers (lateralized movements) for...
Show moreA novel anatomical behavioral descriptive taxonomy improves motion capture in complex motor stereotypies (CMS) by indexing precise time data without degradation in the complexity of whole body movement in CMS. The absence of etiological explanation of complex motor stereotypies warrants the aggregation of a core CMS dataset to compare regulation of repetitive behaviors in the time domain. A set of visual formalisms trap configurations of behavioral markers (lateralized movements) for behavioral phenotype discovery as paired transitions (from, to) and asymmetries within repetitive restrictive behaviors. This translational project integrates NIH MeSH (medical subject headings) taxonomy with direct biological interface (wearable sensors and nanoscience in vitro assays) to design the architecture for exploratory diagnostic instruments. Motion capture technology when calibrated to multi-resolution indexing system (MeSH based) quantifies potential diagnostic criteria for comparing severity of CMS within behavioral plasticity and switching (sustained repetition or cyclic repetition) time-signatures. Diagnostic instruments sensitive to high behavioral resolution promote measurement to maximize behavioral activity while minimizing biological uncertainty. A novel protocol advances CMS research through instruments with recursive design.
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Date Issued: 2015
Identifier: CFE0005927, ucf:50846
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005927

Title: Development of human and rodent based in vitro systems toward better translation of bench to bedside in vivo results.
Creator: Berry, Bonnie, Hickman, James, Khaled, Annette, Lambert, Stephen, Sugaya, Kiminobu, University of Central Florida
Abstract / Description: Prospective medicinal compounds progress through multiple testing phases before becoming licensed drugs. Testing of novel compounds includes a preclinical phase where the potential therapeutic is tested in vitro and/or in animal models in vivo to predict its potential efficacy and/or toxicity in humans. The failure of preclinical models to accurately predict human drug responses can lead to potentially dangerous compounds being administered to humans, or potentially beneficial compounds being...
Show moreProspective medicinal compounds progress through multiple testing phases before becoming licensed drugs. Testing of novel compounds includes a preclinical phase where the potential therapeutic is tested in vitro and/or in animal models in vivo to predict its potential efficacy and/or toxicity in humans. The failure of preclinical models to accurately predict human drug responses can lead to potentially dangerous compounds being administered to humans, or potentially beneficial compounds being kept in development abeyance. Moreover, inappropriate choice in model organism for studying disease states may result in pushing forward inappropriate drug targets and/or compounds and wasting valuable time and resources in producing much-needed medications. In this dissertation, models for basic science research and drug testing are investigated with the intention of improving current preclinical models in order to drive drugs to market faster and more efficiently. We found that embryonic rat hippocampal neurons, commonly used to study neurodegenerative disease mechanisms in vitro, take 3-4 weeks to achieve similar, critical ion-channel expression profiles as seen in adult rat hippocampal cultures. We also characterized a newly-available commercial cell line of human induced pluripotent stem cell-derived neurons for their applicability in long-term studies, and used them to develop a more pathologically relevant model of early Alzheimer's Disease in vitro. Finally, we attempted to create an engineered, layered neural network of human neurons to study drug responses and synaptic mechanisms. Utilization of the results and methods described herein will help push forward the development of better model systems for translation of laboratory research to successful clinical human drug trials.
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Date Issued: 2015
Identifier: CFE0006261, ucf:51031
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006261

Title: IDENTIFICATION OF EPITHELIAL STROMAL INTERACTION 1 AND EPIDERMAL GROWTH FACTOR RECEPTOR AS NOVEL KR(&)#220;PPEL-LIKE FACTOR 8 TARGETS IN PROMOTING BREAST CANCER PROGRESSION.
Creator: Li, Tianshu, Zhao, Jihe, Khaled, Annette, Altomare, Deborah, Lambert, Stephen, University of Central Florida
Abstract / Description: Breast cancer is the major cause of cancer death among women worldwide. Understanding the mechanisms underlying breast cancer progression remains urgent for developing effective treatment strategies to eliminate breast cancer mortality. Our recent studies have demonstrated that Kr(&)#252;ppel-like transcriptional factor 8 (KLF8) plays a critical role for breast cancer progression. Other studies have shown that Epithelial stromal interaction 1 (EPSTI1), a recently identified stromal fibroblast...
Show moreBreast cancer is the major cause of cancer death among women worldwide. Understanding the mechanisms underlying breast cancer progression remains urgent for developing effective treatment strategies to eliminate breast cancer mortality. Our recent studies have demonstrated that Kr(&)#252;ppel-like transcriptional factor 8 (KLF8) plays a critical role for breast cancer progression. Other studies have shown that Epithelial stromal interaction 1 (EPSTI1), a recently identified stromal fibroblast-induced gene in non-invasive breast cancer cells and epidermal growth factor receptor (EGFR) are highly overexpressed in aggressively invasive breast carcinomas including triple negative breast cancers. In this thesis project, we demonstrate high co-overexpression of KLF8 with EPSTI1 as well as EGFR in invasive breast cancer cells and patient tumors. We also show that KLF8 upregulates the expression of EPSTI1 by directly binding and activating the EPSTI1 gene promoter, and KLF8 upregulates the expression of EGFR not only by directly activating the EGFR gene promoter but also by preventing EGFR translation from microRNA141-dependent inhibition. Genetic, signaling and animal cancer model analyses indicate that downstream of KLF8, EPSTI1 promotes the tumor invasion and metastasis by activating NF-?B through binding valosin containing protein (VCP) and subsequent degradation of I?B?, whereas EGFR promotes tumor growth and metastasis via activation of ERK. Taken together, these data identify EPSTI1 and EGFR as novel KLF8 targets in breast cancer and suggest that KLF8 may be targeted for new effective treatment of breast cancer.
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Date Issued: 2013
Identifier: CFE0005366, ucf:50474
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005366

Title: Dissecting the Components of Neuropathic Pain.
Creator: George, Dale, Lambert, Stephen, Kim, Yoon-Seong, Fernandez-Valle, Cristina, Ebert, Steven, University of Central Florida
Abstract / Description: Pain is a public health issue affecting the lives of nearly 116 million adults in the US, annually. Understanding the physiological and phenotypic changes that occur in response to painful stimuli is of tremendous clinical interest, but, the complexity of pain and the lack of a representative in vitro model hinders the development of new therapeutics. Pain stimuli are first perceived and transmitted by the neurons within the dorsal root ganglia (DRG) which become hyperexcitable under these...
Show morePain is a public health issue affecting the lives of nearly 116 million adults in the US, annually. Understanding the physiological and phenotypic changes that occur in response to painful stimuli is of tremendous clinical interest, but, the complexity of pain and the lack of a representative in vitro model hinders the development of new therapeutics. Pain stimuli are first perceived and transmitted by the neurons within the dorsal root ganglia (DRG) which become hyperexcitable under these conditions. It has now been established that satellite glial cells (SGCs) that ensheathe the DRG cell body actively contribute to this neuronal dysregulation. To understand the role of SGCs in this pain circuit, first, we looked at the development of SGCs within the DRG of rats, and we showed that SGCs developed postnatally, and appeared morphologically, transcriptionally and functionally similar to Schwann cells precursors (SCs), supporting the idea that these cells may exhibit multipotent behavior. Secondly, we describe here, a three-dimensional in vitro model of the DRG which is functionally characterized on a microelectrode array (MEA). This model can be used to assess the long-term recording of spontaneous activity from bundles of axons while preserving the neuronal-SGC interactions similar to those observed in vivo. Furthermore, using capsaicin, an agonist of the TRPV1 nociceptive receptor, we show that this model can be used as an in vitro assay to acquire evoked responses from nociceptive neurons. Overall, this study advances our knowledge on the development and differentiation of SGCs and establishes a novel functional three-dimensional model for the study of SGCs. This model can now be used as a tool to study the underlying basis of neuronal dysregulation in pain.
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Date Issued: 2018
Identifier: CFE0007002, ucf:52053
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007002

Title: Development of a Functional In Vitro 3D Model of the Peripheral Nerve.
Creator: Anderson, Wesley, Lambert, Stephen, Hickman, James, Fernandez-Valle, Cristina, Willenberg, Bradley, University of Central Florida
Abstract / Description: Peripheral neuropathies, affect approximately 20 million people in the United States and are often a complication of conditions such as diabetes that can result in amputation of affected areas such as the feet and toes. In vitro methodologies to facilitate the understanding and treatment of these disorders often lack the cellular and functional complexity required to accurately model peripheral neuropathies. In particular, they are often 2-D and functional readouts, such as electrical...
Show morePeripheral neuropathies, affect approximately 20 million people in the United States and are often a complication of conditions such as diabetes that can result in amputation of affected areas such as the feet and toes. In vitro methodologies to facilitate the understanding and treatment of these disorders often lack the cellular and functional complexity required to accurately model peripheral neuropathies. In particular, they are often 2-D and functional readouts, such as electrical activity, are limited to cell bodies thereby limiting the understanding of axonopathy which often characterizes these disorders. We have developed a functional 3-D model of peripheral nerves using a capillary alginate gel (Capgel(TM)), as a scaffold. We hypothesize that: 1) The unique microcapillary structure of Capgel(TM) allows for the modeling of the 3-D microstructure of the peripheral nerve, and 2) That axon bundling in the capillary allows for the detection of axonal electrical activity. In our initial studies, we demonstrate that culturing embryonic dorsal root ganglia (DRG) within the Capgel(TM) environment allows for the separation of cell bodies from axons and recreates many of the features of an in vivo peripheral nerve fascicle including myelinated axons and the formation of a rudimentary perineurium. To develop functionality for this model we have integrated the DRG Capgel(TM) culture with a microelectrode array to examine spontaneous activity in axon bundles, which we find demonstrates superiority to other widely used 2-D models of the same tissue. Furthermore, by analyzing the activity on individual electrodes, we were able to record action potentials from multiple axons within the same bundle indicating a functional complexity comparable to that observed in fascicles in vivo. This 3D model of the peripheral nerve can be used to study the functional complexities of peripheral neuropathies and nerve regeneration as well as being utilized in the development of novel therapeutics.
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Date Issued: 2018
Identifier: CFE0007150, ucf:52303
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007150

Title: The Actin-Severing Protein Cofilin Is Downstream Of Neuregulin Signaling, Is Regulated By The Tumor Suppressor Merlin, And Is Essential For Schwann Cell Myelination.
Creator: Sparrow, Nicklaus, Fernandez-Valle, Cristina, Lambert, Stephen, Ebert, Steven, Altomare, Deborah, University of Central Florida
Abstract / Description: Myelination is a complex process requiring coordination of directional motility and an increase in Schwann cell (SC) size to generate a multi-lamellar myelin sheath. Regulation of actin dynamics during myelination is poorly understood. However, it is known that myelin thickness is related to the abundance of neuregulin1-type III (NRG) expressed on the axon surface. NRG binding to ErbB2/3 receptors on the Schwann cell surface initiates signaling cascades necessary for myelination. We identify...
Show moreMyelination is a complex process requiring coordination of directional motility and an increase in Schwann cell (SC) size to generate a multi-lamellar myelin sheath. Regulation of actin dynamics during myelination is poorly understood. However, it is known that myelin thickness is related to the abundance of neuregulin1-type III (NRG) expressed on the axon surface. NRG binding to ErbB2/3 receptors on the Schwann cell surface initiates signaling cascades necessary for myelination. We identify cofilin1, an actin depolymerizing and severing protein, as a downstream target of NRG-ErbB2/3 signaling in rat SC. A five minute exposure of SCs to NRG triggers phosphorylation of ErbB2 with concomitant dephosphorylation, and activation, of cofilin, and its upstream regulators, LIM domain kinase (LIMK) and Slingshot-1 phosphatase (SSH). This leads to cofilin activation and recruitment to the leading edge of the SC plasma membrane. These changes are associated with rapid plasma membrane expansion yielding a 35(-)50% increase in SC size within 30 minutes of NRG1 exposure. Cofilin1-deficient SCs increase phosphorylation of ErbB2, ERK, focal adhesion kinase, and paxillin in response to NRG, but fail to increase in size possibly due to stabilization of unusually long focal adhesions. Cofilin1-deficient SCs co-cultured with sensory neurons fail to elaborate myelin. Ultrastructural analysis reveals that they unsuccessfully segregate or engage axons and form only patchy basal lamina. After 48 hours of co-culturing with neurons, cofilin-deficient SCs fail to align and elongate on axons and often adhere to the underlying substrate rather than to axons. We show that the Neurofibromatosis Type II (NF2) tumor suppressor, merlin, is an upstream regulator of cofilin1, and that merlin knockdown in Schwann cells inhibits their elaboration of normal myelin sheaths in vitro. Merlin-deficient SCs form shorter myelin segments in DRG neuron/SC co-cultures. Merlin-deficient Schwann cells have increased levels of both active Rac (Rac-GTP) and F-actin indicative of a stable actin cytoskeleton. Surprisingly merlin-deficient Schwann cells fail to dephosphorylate and activate cofilin1 in response to NRG stimulation. Inhibition of LIMK restores the ability of merlin-deficient SCs to activate cofilin in response to NRG. In developing rat sciatic nerve, merlin becomes hyper-phosphorylated at S518 during the time of peak myelin formation. During this time, cofilin is localized to the inner mesaxon, and subsequently to Schmidt-Lanterman incisures in mature myelin. This study: 1) identifies cofilin and its upstream regulators, LIMK and SSH, as end targets of a NRG-ErbB2/3 signaling pathway in Schwann cells, 2) demonstrates that cofilin modulates actin dynamics in Schwann cells allowing for motility needed to effectively engage and myelinate axons, 3) shows that merlin regulates NRG-ErbB2/3-cofilin-actin signaling during SC myelination to determine the myelin segment length.
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Date Issued: 2017
Identifier: CFE0006664, ucf:51217
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006664

Title: Targeted Therapy Development for Neurofibromatosis Type 2.
Creator: Fuse, Marisa, Fernandez-Valle, Cristina, Lambert, Stephen, Altomare, Deborah, Khaled, Annette, University of Central Florida
Abstract / Description: Neurofibromatosis type 2 (NF2) is a debilitating disease characterized by the formation of bilateral vestibular schwannomas, which compress the vestibulocochlear nerve and cause deafness. Additional peripheral schwannomas, meningiomas and ependymomas may also form. NF2 is caused by mutations in the NF2 gene, resulting in the loss of function of the merlin tumor suppressor. Merlin functions in multiple signaling pathways and its absence in Schwann cells yields increased cell survival and...
Show moreNeurofibromatosis type 2 (NF2) is a debilitating disease characterized by the formation of bilateral vestibular schwannomas, which compress the vestibulocochlear nerve and cause deafness. Additional peripheral schwannomas, meningiomas and ependymomas may also form. NF2 is caused by mutations in the NF2 gene, resulting in the loss of function of the merlin tumor suppressor. Merlin functions in multiple signaling pathways and its absence in Schwann cells yields increased cell survival and proliferation, thereby causing schwannoma formation. First line treatment for NF2 is watchful waiting and surgical removal of tumors, potentially resulting in facial paralysis and deafness. To date, there are no pharmacological options for patients with NF2. Since the first clinical trials were completed in 2012, only 5 drugs have been investigated in NF2 patients. Few drugs have elicited a measurable radiographic tumor response and most only result in temporary hearing improvement in a small subset of patients. Development of novel therapeutic compounds is a slow, expensive process. However, re-purposing FDA-approved drugs for NF2 accelerates the transfer of efficacious drugs to the clinic. This dissertation used a systematic approach to identify drugs capable of reducing NF2-associated schwannoma growth. An initial screen revealed drugs that reduced viability of mouse and human merlin-deficient Schwann cells. Efficacious drugs were then advanced to an allograft mouse model of NF2 to identify those that reduced tumor growth in vivo. Drug efficacy was also examined in human primary schwannoma cells. We showed that Src, c-MET and MEK inhibitors reduced viability of merlin-deficient Schwann cells both in vitro and in vivo. We also identified a combination treatment of Src and c-MET inhibitors that induced apoptosis, suggesting the potential for preventing tumor recurrence after completion of drug treatment. The work presented here provides valuable pre-clinical evidence for the advancement of several approved drugs to clinical trials for NF2-associated schwannomas.
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Date Issued: 2017
Identifier: CFE0006877, ucf:51707
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006877

Title: Assessment of molecular interactions via magnetic relaxation: a quest for inhibitors of the anthrax toxin.
Creator: Santiesteban, Oscar, Perez Figueroa, Jesus, Liao, Yi, Yestrebsky, Cherie, Hampton, Michael, Lambert, Stephen, University of Central Florida
Abstract / Description: Anthrax is severe disease caused by the gram-positive Bacillus anthracis that can affect humans with deadly consequences. The disease propagates via the release of bacterial spores that can be naturally found in animals or can be weaponized and intentionally released into the atmosphere in a terrorist attack. Once inhaled, the spores become activated and the anthrax bacterium starts to reproduce and damage healthy macrophages by the release of the anthrax toxin. The anthrax toxin is composed...
Show moreAnthrax is severe disease caused by the gram-positive Bacillus anthracis that can affect humans with deadly consequences. The disease propagates via the release of bacterial spores that can be naturally found in animals or can be weaponized and intentionally released into the atmosphere in a terrorist attack. Once inhaled, the spores become activated and the anthrax bacterium starts to reproduce and damage healthy macrophages by the release of the anthrax toxin. The anthrax toxin is composed of three virulent factors: (i) anthrax protective antigen (APA), (ii) anthrax lethal factor (ALF), and (iii) anthrax edema factor (AEF) that work in harmony to effectuate the lethality associated with the disease. Out of the two internalized factors, ALF has been identified to play a critical role in cell death. Studies in animals have shown that mice infected with an anthrax strain lacking ALF survive the infection whereas when ALF is present the survivability of the mice is eliminated. Although the current therapy for anthrax is antibiotic treatment, modern medicine faces some critical limitations when combating infections. Antibiotics have proven very efficient in eliminating the bacterial infection but they lack the ability to destroy or inhibit the toxins released by the bacteria. This is a significant problem since ALF can remain active in the body for days after the infection is eliminated with no way of inhibiting its destructive effects. The use of inhibitors of ALF is an attractive method to treat the pathogenesis of anthrax infections. Over the last decade several inhibitors of the enzymatic activity of ALF have been identified. In order to identify inhibitors of ALF a variety of screening approaches such as library screenings, Mass Spectroscopy- based screenings and scaffold-based NMR screening have been used. Results from these screening have yielded mainly small molecules that can inhibit ALF in low micromolar to nanomolar concentrations. Yet, although valuable, these results have very little significance with regards to treating ALF in a real-life scenario since pharmaceutical companies are not willing to invest in further developing these inhibitors. Furthermore, the low incidence of inhalation anthrax, the lack of a market for an ALF inhibitor, and the expenses associated with the approval process of the FDA, have hindered the motivation of pharmaceutical companies to pursuit these kind of drugs. Therefore we have screened a small-molecule library of FDA approved drugs and common molecules in order to identify currently approved FDA drugs that can also inhibit ALF (Chapter III). The screening revealed that five molecules: sulindac, fusaric acid, naproxen, ketoprofen and ibuprofen bound to either ALF or APA with sulindac binding both. Additionally, we have developed a nanoparticle-based screening method that assesses molecular interactions by magnetic relaxation changes (Chapter II). Using this assay, we were able to accurately measure the dissociation constants of different interactions between several ligands and macromolecules. Moreover, we have used computational docking studies to predict the binding site of the identified molecules on the ALF or APA (Chapter IV). These studies predicted that two molecules sulindac and fusaric acid could be potential inhibitors of ALF since they bind at the enzymatic pocket. As a result, we tested the inhibitory potential of these molecules as well as that of the metabolic derivatives of sulindac (Chapter V). Results from these studies provided conclusive evidence that fusaric acid and sulindac were both strong inhibitors of ALF. Furthermore, the metabolic derivatives of sulindac, sulindac sulfide and sulindac sulfone also inhibited ALF. Overall, taking together these results we have discovered the alternate use of a currently used drug for the treatment of ALF pathogenesis.
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Date Issued: 2012
Identifier: CFE0004794, ucf:49745
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004794

Title: A high-content multiplexed screening platform for the evaluation and manipulation of force and fatigue of adult derived skeletal muscle myotubes in defined serum-free medium.
Creator: McAleer, Christopher, Hickman, James, Ebert, Steven, Perez Figueroa, J. Manuel, Lambert, Stephen, University of Central Florida
Abstract / Description: The overall focus of this project has two parts: First, was to develop a protocol utilizing serum-free media formulations and defined plating and culture techniques to create functional in vitro myotubes derived from adult skeletal muscle satellite cells. The second was to manipulate the inherent muscle parameters such as force output and fatigue of these myotubes by employing exercise regimes or by small molecule application. The importance of serum-free medium use for in vitro cultures is...
Show moreThe overall focus of this project has two parts: First, was to develop a protocol utilizing serum-free media formulations and defined plating and culture techniques to create functional in vitro myotubes derived from adult skeletal muscle satellite cells. The second was to manipulate the inherent muscle parameters such as force output and fatigue of these myotubes by employing exercise regimes or by small molecule application. The importance of serum-free medium use for in vitro cultures is becoming increasingly important in creating functional systems that can be validated for drug testing by the Food and Drug Administration (FDA). Also, the study of age related diseases as well as the potential for (")personalized medicine(") relies on the proliferation and maturation of satellite cells from adult derived tissue. For that purpose, a serum-free medium and culture system was designed to create mature striated myotubes in culture on a defined non-biological substrate N-1[3-trimethoxysilyl propyl] diethylenetriamine (DETA). These myotubes were evaluated by morphology, muscle specific protein expression, and by muscle functionality. After the thorough characterization of the resultant myotubes the functional output of the muscle was altered utilizing chemical means (creatine supplementation and PGC-1? agonists), chronic long term stimulation, and the use of PGC-1? deficient tissue. In this thesis presentation the utility of the newly developed medium formulation to create myotubes from a variety of adult derived muscle sources will be shown. A protocol in which to exercise skeletal muscle in vitro to alter endurance was developed and employed to manipulate skeletal muscle. Finally, small molecules were tested to validate this system for drug study use. This engineered system has the potential for high-throughput screening of drugs for efficacy and drug toxicity studies as well as general biological studies on muscle fatigue.
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Date Issued: 2015
Identifier: CFE0005660, ucf:50162
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005660

Title: Target validation for Neurofibromatosis Type 2 therapeutics.
Creator: Petrilli Guinart, Alejandra, Fernandez-Valle, Cristina, Altomare, Deborah, Khaled, Annette, Lambert, Stephen, University of Central Florida
Abstract / Description: Neurofibromatosis type 2 (NF2) is a benign tumor disease of the nervous system. Development of bilateral vestibular schwannomas is characteristic of NF2; however patients frequently present schwannomas on other nerves, as well as meningiomas and ependymomas. Currently, there are no drug therapies for NF2. There is an urgent need for development of NF2 therapeutics and this dissertation presents two independent potential therapeutic targets.The disease is caused by mutations in the NF2 gene...
Show moreNeurofibromatosis type 2 (NF2) is a benign tumor disease of the nervous system. Development of bilateral vestibular schwannomas is characteristic of NF2; however patients frequently present schwannomas on other nerves, as well as meningiomas and ependymomas. Currently, there are no drug therapies for NF2. There is an urgent need for development of NF2 therapeutics and this dissertation presents two independent potential therapeutic targets.The disease is caused by mutations in the NF2 gene that encodes a tumor suppressor called merlin. Loss of merlin function is associated with increased activity of Rac and p21-activated kinases (PAK) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrates for Cdc42/Rac-PAK, and modulate actin dynamics by phosphorylating cofilin, an actin severing and depolymerizing agent. LIMKs also translocate into the nucleus and regulate cell cycle progression. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2 delta Ex2) exhibited increased levels of LIMK1, LIMK2, and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared to wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared to normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2?delta Ex2 MSC reduced LIMK1 and LIMK2 levels. Pharmacological inhibition of LIMK with BMS-5, decreased the viability of Nf2?delta Ex2 MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2?delta Ex2 MSCs. The decreased viability of Nf2?delta Ex2 MSCs was due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrest cells in early mitosis by decreasing Aurora A activation and cofilin phosphorylation.To increase the search for NF2 therapeutics, we applied an alternative approach to drug discovery with an unbiased pilot high-throughput screen of the Library of Pharmacologically Active Compounds. We assayed for compounds capable of reducing viability of Nf2?delta Ex2 MSC as a cellular model for human NF2 schwannomas. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2, a mammalian sirtuin, is a NAD+-dependent protein deacetylase. We show that Nf2?delta Ex2 MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases Nf2?delta Ex2 MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability. Inhibition of SIRT2 activity in Nf2?delta Ex2 MSC causes cell death accompanied by release of the necrotic markers lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest.Overall this work uncovered two novel potential therapeutic targets, LIMK and SIRT2 for NF2 and tumors associated with merlin deficiency.
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Date Issued: 2013
Identifier: CFE0005398, ucf:50453
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005398

Title: Role of Cardiac Catecholamines in Embryos and Adults Under Stress.
Creator: Baker, Candice, Ebert, Steven, Bossy-Wetzel, Ella, Siddiqi, Shadab, Lambert, Stephen, University of Central Florida
Abstract / Description: Cardiovascular disease is responsible for the loss of one life every 38 seconds and accounts for 26.6 percent of all infants that die of congenital birth defects. Adrenergic hormones are critically important regulators of cardiovascular physiology in embryos and adults. They are key mediators of stress responses and have profound stimulatory effects on cardiovascular function, and dysregulation of adrenergic function has been associated with many adverse cardiac conditions, including...
Show moreCardiovascular disease is responsible for the loss of one life every 38 seconds and accounts for 26.6 percent of all infants that die of congenital birth defects. Adrenergic hormones are critically important regulators of cardiovascular physiology in embryos and adults. They are key mediators of stress responses and have profound stimulatory effects on cardiovascular function, and dysregulation of adrenergic function has been associated with many adverse cardiac conditions, including congenital malformations, arrhythmias, ischemic heart disease, heart failure, and sudden cardiac death. Despite intensive study, the specific roles these hormones play in the developing heart is not well-understood. Further, there is little information available regarding how these important hormones mediate stress responses in adult females (before and after menopause) in comparison to males. My thesis thus has two major foci: (1) What role(s) do catecholamines play in the embryonic heart?, and (2) Do catecholamines differentially influence cardiac function in aging male and female hearts? Initially, we sought to uncover the roles of adrenergic hormones in the embryonic heart by utilizing an adrenergic-deficient (Dbh-/-) mouse model. We found that adrenergic hormones influence heart development by stimulating expression of the gap junction protein, connexin 43, facilitating atrioventricular conduction, and helping to maintain cardiac rhythm. As development progresses, cardiac energy demands increase substantially, and oxidative phosphorylation becomes vital. Adrenergic hormones regulate metabolism in adults, thus we hypothesized they may stimulate energy metabolism during the embryonic/fetal transition period. We examined ATP, ADP, oxygen consumption rate, and extracellular acidification rates and found these metabolic indices were significantly decreased in Dbh-/- hearts compared to Dbh+/+ controls. We employed transmission electron microscopy of embryonic cardiomyocytes and found the mitochondria were significantly larger in Dbh-/- hearts compared to controls, and had more branch points. Taken together, these results suggest adrenergic hormones play a major role mediating the shift from predominantly anaerobic to aerobic metabolism during the embryonic/fetal transition period.Since there are known differential cardiac responses due to sex, age, and menopause to stress, we used echocardiography to measure left ventricular (LV) function in adult (9, 18 and 21 month) male and female mice (pre and postmenopausal) in response to epinephrine, and immobilization stress to investigate the roles of these factors. My results show 9-month premenopausal female mice display significantly decreased LV responsiveness to epinephrine compared to males, and an increased response to epinephrine due to age, especially in the premenopausal females. Similar LV function was also observed between postmenopausal females and males, and this pattern persisted after immobilization stress. I also investigated anatomical differences in the distribution of adrenergic cells within the heart comparing age, sex, and menopausal status. Notably, the density of cells derived from an adrenergic lineage in the heart was significantly increased in postmenopausal mice compared to age-matched males and cycling females. The selective re-appearance of adrenergic cells in the heart following menopause may provide an explanation for the differential stress responses observed in our system, and could have important clinical ramifications for stress-induced cardiomyopathies.
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Date Issued: 2014
Identifier: CFE0005458, ucf:50373
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005458

Schwann cell (3)	+ -
microelectrode array (3)	+ -
body-on-a-chip (2)	+ -
electrophysiology (2)	+ -
merlin (2)	+ -

3D model (1)	+ -
3D printing (1)	+ -
Adrenergic Hormones (1)	+ -
Alzheimer's (1)	+ -
BzATP (1)	+ -
Cardiomyopathy (1)	+ -
Cardiovascular (1)	+ -
Development (1)	+ -
EGFR (1)	+ -
EPSTI1 (1)	+ -
Exercise (1)	+ -
Glia (1)	+ -
High-Content (1)	+ -
KLF8 (1)	+ -
LIMK (1)	+ -
MEA (1)	+ -
Microelectrode array (1)	+ -
Myelin (1)	+ -
NF2 (1)	+ -
Nerve guide (1)	+ -
Nerve regeneration (1)	+ -
Neurofibromatosis Type 2 (1)	+ -
Neuron (1)	+ -
P2X7 (1)	+ -
Perineurium (1)	+ -

ETD Collection (13)	+ -
Honors Collection (1)	+ -

University of Central Florida (14)	+ -
Lambert, Stephen (13)	+ -
Ebert, Steven (5)	+ -
Fernandez-Valle, Cristina (5)	+ -
Altomare, Deborah (4)	+ -

Hickman, James (4)	+ -
Khaled, Annette (4)	+ -
Kim, Yoon-Seong (2)	+ -
Anderson, Wesley (1)	+ -
Aubin, Megan (1)	+ -
Baker, Candice (1)	+ -
Berry, Bonnie (1)	+ -
Bobbitt, Nathaniel (1)	+ -
Bossy-Wetzel, Ella (1)	+ -
Coffey, Kevin (1)	+ -
Fuse, Marisa (1)	+ -
George, Dale (1)	+ -
Hampton, Michael (1)	+ -
Hughes, Charles (1)	+ -
Kursewicz, Christina D (1)	+ -
Lambert,Stephen (1)	+ -
Li, Tianshu (1)	+ -
Liao, Yi (1)	+ -
Manja, Sanjana (1)	+ -
McAleer, Christopher (1)	+ -
Perez Figueroa, J. Manuel (1)	+ -
Perez Figueroa, Jesus (1)	+ -
Petrilli Guinart, Alejandra (1)	+ -
Santiesteban, Oscar (1)	+ -
Siddiqi, Shadab (1)	+ -

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