Matching Items (40)

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Development of a Novel Zebrafish Model for Dynamin-1 Epileptic Encephalopathy

Description

Epileptic encephalopathies (EE) are genetic or environmentally-caused conditions that cause “catastrophic” damage or degradation to the sensory, cognitive, and behavioral centers of the brain. Whole-exome sequencing identified de novo heterozygous missense mutations within the DNM1 gene of five pediatric patients

Epileptic encephalopathies (EE) are genetic or environmentally-caused conditions that cause “catastrophic” damage or degradation to the sensory, cognitive, and behavioral centers of the brain. Whole-exome sequencing identified de novo heterozygous missense mutations within the DNM1 gene of five pediatric patients with epileptic encephalopathies. DNM1 encodes for the dynamin-1 protein which is involved in endocytosis and synaptic recycling, and it is a member of dynamin GTPase. The zebrafish, an alternative model system for drug discovery, was utilized to develop a novel model for dynamin-1 epileptic encephalopathy through a small molecule inhibitor. The model system mimicked human epilepsy caused by DNM1 mutations and identified potential biochemical pathways involved in the production of this phenotype. The use of microinjections of mutated DNM1 verified phenotypes and was utilized to determine safe and effective antiepileptic drugs (AEDs) for treatment of this specific EE. This zebrafish dynamin-1 epileptic encephalopathy model has potential uses for drug discovery and investigation of this rare childhood disorder.

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Date Created
2019-05

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Optimization of Human Neural Tissue Clearing for Immunohistochemical Imaging

Description

The combination of immunohistochemical (IHC) stainings and optical microscopy has allowed for the visualization of specific microscopic structures within tissue; however, limitations in light and antibody penetration mitigate the scale on which these images can be taken (Alshammari et al,

The combination of immunohistochemical (IHC) stainings and optical microscopy has allowed for the visualization of specific microscopic structures within tissue; however, limitations in light and antibody penetration mitigate the scale on which these images can be taken (Alshammari et al, 2016; Marx, 2014). Tissue clearing, specifically the removal of lipids to improve sample transparency, solves the former weakness well, but does not improve antibody penetration significantly (Chung et al, 2013; Treweek et al, 2015). Therefore, there is a need to equalize the maximum depth that light can pass through a section with the depth at which there is recognizable fluorescence. This is particularly important when staining blood vessels as traditional size limitations exclusively allows for cross sectional visualization. Passive CLARITY Technique (PACT) has been at the forefront of tissue clearing protocols, utilizing an acrylamide hydrogel solution to maintain structure and sodium dodecyl sulfate to wash out lipids (Tomer et al, 2014). PACT is limited in its ability to clear larger sections and is not conducive to IHC antibody diffusion (Treweek et al, 2015). In order to circumvent these drawbacks, CUBIC was developed as an alternative passive protocol, aimed at being scalable to any tissue size (Richardson, 2015; Susaki et al, 2015). This study compared the effectiveness of both protocols in high and low lipid tissues in the context of blood vessel staining efficacy. Upon initial comparison, it became apparent that there was a statistically significant difference in mean DAPI intensity at all depths, up to 200 micrometers, between CUBIC and PACT \u2014 the former showcasing brighter stainings. Moreover, it was found that PACT does not remove erythrocytes from the tissue meaning that their auto-fluorescence is seen during imaging. Therefore, for blood vessel stainings, only CUBIC was optimized and quantitatively analyzed. In both tissue conditions as well as for two stainings, DAPI and fibronectin (FNCT), optimized CUBIC demonstrated a statistically significant difference from standard CUBIC with regards to mean fluorescent intensity.

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Date Created
2018-05

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Direct nose-to-brain delivery of targeted polymeric nanoparticles

Description

There is growing interest in intranasal delivery of therapeutics because of direct nose-to-brain pathways which are able to bypass biological barriers, such as the blood-brain barrier (BBB), that have historically limited our ability to effectively deliver drugs to the central

There is growing interest in intranasal delivery of therapeutics because of direct nose-to-brain pathways which are able to bypass biological barriers, such as the blood-brain barrier (BBB), that have historically limited our ability to effectively deliver drugs to the central nervous system (CNS). Since these pathways were first discovered, there has been significant preclinical success in delivering a wide range of therapeutics to the CNS with additional growing efforts to further improve delivery through nanoparticle drug delivery systems. Here we sought to improve intranasal delivery of DiR, a lipophilic small molecule cyanine dye, to the CNS by surface modifying a poly (lactic-co-glycolic acid) (PLGA) nanoparticle with a short peptide derived from the rabies virus glycoprotein (RVG). The specific aims of this thesis were to evaluate administration route-dependent delivery of RVG nanoparticles to the CNS, and to identify anatomical transport pathways by which nanoparticles facilitate transport of small lipophilic molecules. Route-dependent delivery kinetics and distribution were studied by administering DiR loaded nanoparticles to healthy Balb/C mice. Specific tissues were homogenized and the fluorescent intensity of DiR was measured and compared to control tissue spiked with known amounts of dye. While bioavailability of DiR after intranasal administration was near 0% with minimal exposure to peripheral organs, quick and efficient delivery to the CNS was still observed. CNS delivery after intranasal administration was rapid with peak concentrations at 30 minutes post-administration followed by broad clearance by 2 hours. Regional differences of delivery of DiR to the CNS demonstrated engagement of direct nose-to-brain transport pathways with high delivery being observed to the olfactory bulb, brain stem, and trigeminal nerve. RVG modification however presented only modest targeting benefits. In conclusion, the biodistribution of DiR after intranasal administration of DiR loaded nanoparticles showed high potential for the direct nose-to-brain delivery while limiting peripheral exposure of lipophilic small molecule drugs.

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Created

Date Created
2016-05

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Development of a Novel Smart Contrast Agent for Magnetic Resonance Imaging

Description

Smart contrast agents allow for noninvasive study of specific events or tissue conditions inside of a patient's body using Magnetic Resonance Imaging (MRI). This research aims to develop and characterize novel smart contrast agents for MRI that respond to temperature

Smart contrast agents allow for noninvasive study of specific events or tissue conditions inside of a patient's body using Magnetic Resonance Imaging (MRI). This research aims to develop and characterize novel smart contrast agents for MRI that respond to temperature changes in tissue microenvironments. Transmission Electron Microscopy, Nuclear Magnetic Resonance, and cell culture growth assays were used to characterize the physical, magnetic, and cytotoxic properties of candidate nanoprobes. The nanoprobes displayed thermosensitve MR properties with decreasing relaxivity with temperature. Future work will be focused on generating and characterizing photo-active analogues of the nanoprobes that could be used for both treatment of tissues and assessment of therapy.

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Created

Date Created
2014-05

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Utilization of Nanoparticles for Identifying Fibrin Deposition in Neural Tissue

Description

The main objective of this research is to develop and characterize a targeted contrast agent that will recognize acute neural injury pathology (i.e. fibrin) after traumatic brain injury (TBI). Single chain fragment variable antibodies (scFv) that bind specifically to fibrin

The main objective of this research is to develop and characterize a targeted contrast agent that will recognize acute neural injury pathology (i.e. fibrin) after traumatic brain injury (TBI). Single chain fragment variable antibodies (scFv) that bind specifically to fibrin have been produced and purified. DSPE-PEG micelles have been produced and the scFv has been conjugated to the surface of the micelles; this nanoparticle system will be used to overcome limitations in diagnosing TBI. The binding and imaging properties will be analyzed in the future to determine functionality of the nanoparticle system in vivo.

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Created

Date Created
2014-05

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Improving the Realism and Magnetic Resonance Imaging of Multicellular Tumor Spheroids

Description

Magnetic resonance imaging (MRI) of changes in metabolic activity in tumors and metabolic abnormalities can provide a window to understanding the complex behavior of malignant tumors. Both diagnostics and treatment options can be improved through the further comprehension of the

Magnetic resonance imaging (MRI) of changes in metabolic activity in tumors and metabolic abnormalities can provide a window to understanding the complex behavior of malignant tumors. Both diagnostics and treatment options can be improved through the further comprehension of the processes that contribute to tumor malignancy and growth. By detecting and disturbing this activity through personalized treatments, it is the hope to provide better diagnostics and care to patients. Experimenting with multicellular tumor spheroids (MCTS) allows for a rapid, inexpensive and convenient solution to studying multiple in vitro tumors. High quality magnetic resonance images of small samples, such as spheroid, however, are difficult to achieve with current radio frequency coils. In addition, in order for the information provided by these scans to accurately represent the interactions and metabolic activity in vivo, there is a need for a perfused vascular network. A perfused vascular network has the potential to improve metabolic realism and particle transport within a tumor spheroid. By creating a more life-like cancer model and allowing the progressive imaging of metabolic functions of such small samples, a better, more efficient mode of studying metabolic activity in cancer can be created and research efforts can expand. The progress described in this paper attempts to address both of these current shortcomings of metabolic cancer research and offers potential solutions, while acknowledging the potential of future work to improve cancer research with MCTS.

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Date Created
2016-12

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A Novel Computing Platform for Accelerated Magnetic Resonance Spectroscopic Cancer Imaging

Description

Compressed sensing magnetic resonance spectroscopic imaging (MRSI) is a noninvasive and in vivo potential diagnostic technique for cancer imaging. This technique undersamples the distribution of specific cancer biomarkers within an MR image as well as changes in the temporal dimension

Compressed sensing magnetic resonance spectroscopic imaging (MRSI) is a noninvasive and in vivo potential diagnostic technique for cancer imaging. This technique undersamples the distribution of specific cancer biomarkers within an MR image as well as changes in the temporal dimension and subsequently reconstructs the missing data. This technique has been shown to retain a high level of fidelity even with an acceleration factor of 5. Currently there exist several different scanner types that each have their separate analytical methods in MATLAB. A graphical user interface (GUI) was created to facilitate a single computing platform for these different scanner types in order to improve the ease and efficiency with which researchers and clinicians interact with this technique. A GUI was successfully created for both prospective and retrospective MRSI data analysis. This GUI retained the original high fidelity of the reconstruction technique and gave the user the ability to load data, load reference images, display intensity maps, display spectra mosaics, generate a mask, display the mask, display kspace and save the corresponding spectra, reconstruction, and mask files. Parallelization of the reconstruction algorithm was explored but implementation was ultimately unsuccessful. Future work could consist of integrating this parallelization method, adding intensity overlay functionality and improving aesthetics.

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Created

Date Created
2016-05

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A Novel Temperature-Sensitive MR & Fluorescence Imaging Contrast Agent

Description

Contrast agents in medical imaging can help visualize structural details, distributions of particular cell types, or local environment characteristics. Multi-modal imaging techniques have become increasingly popular for their improved sensitivity, resolution, and ability to correlate structural and functional information. This

Contrast agents in medical imaging can help visualize structural details, distributions of particular cell types, or local environment characteristics. Multi-modal imaging techniques have become increasingly popular for their improved sensitivity, resolution, and ability to correlate structural and functional information. This study addresses the development of dual-modality (magnetic resonance/fluorescence) and dual-functional (thermometry/detection) nanoprobes for enhanced tissue imaging.

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Date Created
2015-05

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Temporal Assessment of Nanoparticle Accumulation After Experimental Brain Injury: Effect of Particle Size

Description

Nanoparticle (NP) based therapeutic and theranostic agents have been developed for various diseases, yet application to neural disease/injury is restricted by the blood-brain-barrier (BBB). Traumatic brain injury (TBI) results in a host of pathological alterations, including transient breakdown of the

Nanoparticle (NP) based therapeutic and theranostic agents have been developed for various diseases, yet application to neural disease/injury is restricted by the blood-brain-barrier (BBB). Traumatic brain injury (TBI) results in a host of pathological alterations, including transient breakdown of the BBB, thus opening a window for NP delivery to the injured brain tissue. This study focused on investigating the spatiotemporal accumulation of different sized NPs after TBI. Specifically, animal cohorts sustaining a controlled cortical impact injury received an intravenous injection of PEGylated NP cocktail (20, 40, 100, and 500 nm, each with a unique fluorophore) immediately (0 h), 2 h, 5 h, 12 h, or 23 h after injury. NPs were allowed to circulate for 1 h before perfusion and brain harvest. Confocal microscopy demonstrated peak NP accumulation within the injury penumbra 1 h post-injury. An inverse relationship was found between NP size and their continued accumulation within the penumbra. NP accumulation preferentially occurred in the primary motor and somatosensory areas of the injury penumbra as compared to the parietal association and visual area. Thus, we characterized the accumulation of particles up to 500 nm at different times acutely after injury, indicating the potential of NP-based TBI theranostics in the acute period after injury.

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Date Created
2016-07-22

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Modeling and Characterization of Mass Transfer Kinetics in Tumor Tissue Using Dynamic Contrast Enhanced Magnetic Resonance Imaging (DCE-MRI)

Description

The objective of the research presented here was to validate the use of kinetic models for the analysis of the dynamic behavior of a contrast agent in tumor tissue and evaluate the utility of such models in determining kinetic properties

The objective of the research presented here was to validate the use of kinetic models for the analysis of the dynamic behavior of a contrast agent in tumor tissue and evaluate the utility of such models in determining kinetic properties - in particular perfusion and molecular binding uptake associated with tissue hypoxia - of the imaged tissue, from concentration data acquired with dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) procedure. Data from two separate DCE-MRI experiments, performed in the past, using a standard contrast agent and a hypoxia-binding agent respectively, were analyzed. The results of the analysis demonstrated that the models used may provide novel characterization of the tumor tissue properties. Future research will work to further characterize the physical significance of the estimated parameters, particularly to provide quantitative oxygenation data for the imaged tissue.

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Created

Date Created
2013-12