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Image Analysis for Registration, Segmentation, and Intensity Measurement of 2-Photon Microscope Images using MATLAB

Description

Traumatic brain injury (TBI) is a major concern in public health due to its prevalence and effect. Every year, about 1.7 million TBIs are reported [7]. According to the According to the Centers for Disease Control and Prevention (CDC), 5.5%

Traumatic brain injury (TBI) is a major concern in public health due to its prevalence and effect. Every year, about 1.7 million TBIs are reported [7]. According to the According to the Centers for Disease Control and Prevention (CDC), 5.5% of all emergency department visits, hospitalizations, and deaths from 2002 to 2006 are due to TBI [8]. The brain's natural defense, the Blood Brain Barrier (BBB), prevents the entry of most substances into the brain through the blood stream, including medicines administered to treat TBI [11]. TBI may cause the breakdown of the BBB, and may result in increased permeability, providing an opportunity for NPs to enter the brain [3,4]. Dr. Stabenfeldt's lab has previously established that intravenously injected nanoparticles (NP) will accumulate near the injury site after focal brain injury [4]. The current project focuses on confirmation of the accumulation or extravasation of NPs after brain injury using 2-photon microscopy. Specifically, the project used controlled cortical impact injury induced mice models that were intravenously injected with 40nm NPs post-injury. The MATLAB code seeks to analyze the brain images through registration, segmentation, and intensity measurement and evaluate if fluorescent NPs will accumulate in the extravascular tissue of injured mice models. The code was developed with 2D bicubic interpolation, subpixel image registration, drawn dimension segmentation and fixed dimension segmentation, and dynamic image analysis. A statistical difference was found between the extravascular tissue of injured and uninjured mouse models. This statistical difference proves that the NPs do extravasate through the permeable cranial blood vessels in injured cranial tissue.

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2018-05

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Pharmacologic Modulation of the Blood-Brain Barrier

Description

One of the most prominent biological challenges for the field of drug delivery is the blood-brain barrier. This physiological system blocks the entry of or actively removes almost all small molecules into the central nervous system (CNS), including many drugs

One of the most prominent biological challenges for the field of drug delivery is the blood-brain barrier. This physiological system blocks the entry of or actively removes almost all small molecules into the central nervous system (CNS), including many drugs that could be used to treat diseases in the CNS. Previous studies have shown that activation of the adenosine receptor signaling pathway through the use of agonists has been demonstrated to increase BBB permeability. For example, regadenoson is an adenosine A2A receptor agonist that has been shown to disrupt the BBB and allow for increased drug uptake in the CNS. The goal of this study was to verify this property of regadenoson. We hypothesized that co-administration of regadenoson with a non-brain penetrant macromolecule would facilitate its entry into the central nervous system. To test this hypothesis, healthy mice were administered regadenoson or saline concomitantly with a fluorescent dextran solution. The brain tissue was either homogenized to measure quantity of fluorescent molecule, or cryosectioned for imaging with confocal fluorescence microscopy. These experiments did not identify any significant difference in the amount of fluorescence detected in the brain after regadenoson treatment. These results contradict those of previous studies and highlight potential differences in injection methodology, time windows, and properties of brain impermeant molecules.

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2015-05

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The Effect of Nanoparticle Diameter on TAT-mediated Delivery to the CNS In Vivo

Description

Neurological disorders are difficult to treat with current drug delivery methods due to their inefficiency and the lack of knowledge of the mechanisms behind drug delivery across the blood brain barrier (BBB). Nanoparticles (NPs) are a promising drug delivery method

Neurological disorders are difficult to treat with current drug delivery methods due to their inefficiency and the lack of knowledge of the mechanisms behind drug delivery across the blood brain barrier (BBB). Nanoparticles (NPs) are a promising drug delivery method due to their biocompatibility and ability to be modified by cell penetrating peptides, such as transactivating transciptor (TAT) peptide, which has been shown to increase efficiency of delivery. There are multiple proposed mechanisms of TAT-mediated delivery that also have size restrictions on the molecules that can undergo each BBB crossing mechanism. The effect of nanoparticle size on TAT-mediated delivery in vivo is an important aspect to research in order to better understand the delivery mechanisms and to create more efficient NPs. NPs called FluoSpheres are used because they come in defined diameters unlike polymeric NPs that have a broad distribution of diameters. Both modified and unmodified 100nm and 200nm NPs were able to bypass the BBB and were seen in the brain, spinal cord, liver, and spleen using confocal microscopy and a biodistribution study. Statistically significant differences in delivery rate of the different sized NPs or between TAT-modified and unmodified NPs were not found. Therefore in future work a larger range of diameter size will be evaluated. Also the unmodified NPs will be conjugated with scrambled peptide to ensure that both unmodified and TAT-modified NPs are prepared in identical fashion to better understand the role of size on TAT targeting. Although all the NPs were able to bypass the BBB, future work will hopefully provide a better representation of how NP size effects the rate of TAT-mediated delivery to the CNS.

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2016-05

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Novel siloxane nanoprobes for molecular and cellular imagin

Description

Oxygen delivery is crucial for the development of healthy, functional tissue. Low tissue oxygenation, or hypoxia, is a characteristic that is common in many tumors. Hypoxia contributes to tumor malignancy and can reduce the success of chemotherapy and radiation treatment.

Oxygen delivery is crucial for the development of healthy, functional tissue. Low tissue oxygenation, or hypoxia, is a characteristic that is common in many tumors. Hypoxia contributes to tumor malignancy and can reduce the success of chemotherapy and radiation treatment. There is a current need to noninvasively measure tumor oxygenation or pO2 in patients to determine a personalized treatment method. This project focuses on creating and characterizing nanoemulsions using a pO2 reporter molecule hexamethyldisiloxane (HMDSO) and its longer chain variants as well as assessing their cytotoxicity. We also explored creating multi-modal (MRI/Fluorescence) nanoemulsions.

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2013-05

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Antibody based diagnostic and therapeutic approach for Alzheimer's disease

Description

Alzheimer's disease (AD) is the most common form of dementia leading to cognitive dysfunction and memory loss as well as emotional and behavioral disorders. It is the 6th leading cause of death in United States, and the only one among

Alzheimer's disease (AD) is the most common form of dementia leading to cognitive dysfunction and memory loss as well as emotional and behavioral disorders. It is the 6th leading cause of death in United States, and the only one among top 10 death causes that cannot be prevented, cured or slowed. An estimated 5.4 million Americans live with AD, and this number is expected to triple by year 2050 as the baby boomers age. The cost of care for AD in the US is about $200 billion each year. Unfortunately, in addition to the lack of an effective treatment or AD, there is also a lack of an effective diagnosis, particularly an early diagnosis which would enable treatment to begin before significant neuronal damage has occurred.

Increasing evidence implicates soluble oligomeric forms of beta-amyloid and tau in the onset and progression of AD. While many studies have focused on beta-amyloid, soluble oligomeric tau species may also play an important role in AD pathogenesis. Antibodies that selectively identify and target specific oligomeric tau variants would be valuable tools for both diagnostic and therapeutic applications and also to study the etiology of AD and other neurodegenerative diseases.

Recombinant human tau (rhTau) in monomeric, dimeric, trimeric and fibrillar forms were synthesized and purified to perform LDH assay on human neuroblastoma cells, so that trimeric but not monomeric or dimeric rhTau was identified as extracellularly neurotoxic to neuronal cells. A novel biopanning protocol was designed based on phage display technique and atomic force microscopy (AFM), and used to isolate single chain antibody variable domain fragments (scFvs) that selectively recognize the toxic tau oligomers. These scFvs selectively bind tau variants in brain tissue of human AD patients and AD-related tau transgenic rodent models and have potential value as early diagnostic biomarkers for AD and as potential therapeutics to selectively target toxic tau aggregates.

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2014

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Novel nuclear magnetic resonance coil for magnetic resonance mesoscopy

Description

Magnetic Resonance Imaging (MRI) is an efficient non-invasive imaging tool widely used in medical field to produce high quality images. The MRI signal is detected with specifically developed radio frequency (RF) systems or "coils". There are several key parameters to

Magnetic Resonance Imaging (MRI) is an efficient non-invasive imaging tool widely used in medical field to produce high quality images. The MRI signal is detected with specifically developed radio frequency (RF) systems or "coils". There are several key parameters to evaluate the performance of RF coils: signal-to-noise ratio (SNR), homogeneity, quality factor (Q factor), sensitivity, etc. The choice of coil size and configuration depends on the object to be imaged. While surface coils have better sensitivity, volume coils are often employed to image a larger region of interest (ROI) as they display better spatial homogeneity. For the cell labeling and imaging studies using the newly developed siloxane based nanoemulsions as 1H MR reporter probes, the first step is to determine the sensitivity of signal detection under controlled conditions in vitro. In this thesis, a novel designed 7 Tesla RF volume coil was designed and tested for detection of small quantities of siloxane probe as well as for imaging of labeled tumor spheroid. The procedure contains PCB circuit design, RF probe design, test and subsequent modification. In this report, both theory and design methodology will be discussed.

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2014

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High-throughput platforms for tumor dormancy-relapse and biomolecule binding using aminoglycoside-derived hydrogels

Description

Relapse after tumor dormancy is one of the leading causes of cancer recurrence that ultimately leads to patient mortality. Upon relapse, cancer manifests as metastases that are linked to almost 90% cancer related deaths. Capture of the dormant and relapsed

Relapse after tumor dormancy is one of the leading causes of cancer recurrence that ultimately leads to patient mortality. Upon relapse, cancer manifests as metastases that are linked to almost 90% cancer related deaths. Capture of the dormant and relapsed tumor phenotypes in high-throughput will allow for rapid targeted drug discovery, development and validation. Ablation of dormant cancer will not only completely remove the cancer disease, but also will prevent any future recurrence. A novel hydrogel, Amikagel, was developed by crosslinking of aminoglycoside amikacin with a polyethylene glycol crosslinker. Aminoglycosides contain abundant amount of easily conjugable groups such as amino and hydroxyl moieties that were crosslinked to generate the hydrogel. Cancer cells formed 3D spheroidal structures that underwent near complete dormancy on Amikagel high-throughput drug discovery platform. Due to their dormant status, conventional anticancer drugs such as mitoxantrone and docetaxel that target the actively dividing tumor phenotype were found to be ineffective. Hypothesis driven rational drug discovery approaches were used to identify novel pathways that could sensitize dormant cancer cells to death. Strategies were used to further accelerate the dormant cancer cell death to save time required for the therapeutic outcome.

Amikagel’s properties were chemo-mechanically tunable and directly impacted the outcome of tumor dormancy or relapse. Exposure of dormant spheroids to weakly stiff and adhesive formulation of Amikagel resulted in significant relapse, mimicking the response to changes in extracellular matrix around dormant tumors. Relapsed cells showed significant differences in their metastatic potential compared to the cells that remained dormant after the induction of relapse. Further, the dissertation discusses the use of Amikagels as novel pDNA binding resins in microbead and monolithic formats for potential use in chromatographic purifications. High abundance of amino groups allowed their utilization as novel anion-exchange pDNA binding resins. This dissertation discusses Amikagel formulations for pDNA binding, metastatic cancer cell separation and novel drug discovery against tumor dormancy and relapse.

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Date Created
2016

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Targeting astrogliosis: isolation and characterization of astrocyte specific single chain antibody fragments

Description

Specificity and affinity towards a given ligand/epitope limit target-specific delivery. Companies can spend between $500 million to $2 billion attempting to discover a new drug or therapy; a significant portion of this expense funds high-throughput screening to find the most

Specificity and affinity towards a given ligand/epitope limit target-specific delivery. Companies can spend between $500 million to $2 billion attempting to discover a new drug or therapy; a significant portion of this expense funds high-throughput screening to find the most successful target-specific compound available. A more recent addition to discovering highly specific targets is the application of phage display utilizing single chain variable fragment antibodies (scFv). The aim of this research was to employ phage display to identify pathologies related to traumatic brain injury (TBI), particularly astrogliosis. A unique biopanning method against viable astrocyte cultures activated with TGF-β achieved this aim. Four scFv clones of interest showed varying relative affinities toward astrocytes. One of those four showed the ability to identify reactive astroctyes over basal astrocytes through max signal readings, while another showed a statistical significance in max signal reading toward basal astrocytes. Future studies will include further affinity characterization assays. This work contributes to the development of targeting therapeutics and diagnostics for TBI.

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Date Created
2013

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Siloxane based cellular labeling: functional applications in 1H MRI

Description

Modern medical conditions, including cancer, traumatic brain injury, and cardiovascular disease, have elicited the need for cell therapies. The ability to non-invasively track cells in vivo in order to evaluate these therapies and explore cell dynamics is necessary. Magnetic Resonance

Modern medical conditions, including cancer, traumatic brain injury, and cardiovascular disease, have elicited the need for cell therapies. The ability to non-invasively track cells in vivo in order to evaluate these therapies and explore cell dynamics is necessary. Magnetic Resonance Imaging provides a platform to track cells as a non-invasive modality with superior resolution and soft tissue contrast. A new methodology for cellular labeling and imaging uses Nile Red doped hexamethyldisiloxane (HMDSO) nanoemulsions as dual modality (Magnetic Resonance Imaging/Fluorescence), dual-functional (oximetry/ detection) nanoprobes. While Gadolinium chelates and super paramagnetic iron oxide-based particles have historically provided contrast enhancement in MRI, newer agents offer additional advantages. A technique using 1H MRI in conjunction with an oxygen reporter molecule is one tool capable of providing these benefits, and can be used in neural progenitor cell and cancer cell studies. Proton Imaging of Siloxanes to Map Tissue Oxygenation Levels (PISTOL) provides the ability to track the polydimethylsiloxane (PDMS) labeled cells utilizing the duality of the nanoemulsions. 1H MRI based labeling of neural stem cells and cancer cells was successfully demonstrated. Additionally, fluorescence labeling of the nanoprobes provided validation of the MRI data and could prove useful for quick in vivo verification and ex vivo validation for future studies.

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Date Created
2014

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A robust vitronectin-derived peptide substrate for the scalable long-term expansion and neuronal differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (hNPCs)

Description

Several debilitating neurological disorders, such as Alzheimer's disease, stroke, and spinal cord injury, are characterized by the damage or loss of neuronal cell types in the central nervous system (CNS). Human neural progenitor cells (hNPCs) derived from human pluripotent stem

Several debilitating neurological disorders, such as Alzheimer's disease, stroke, and spinal cord injury, are characterized by the damage or loss of neuronal cell types in the central nervous system (CNS). Human neural progenitor cells (hNPCs) derived from human pluripotent stem cells (hPSCs) can proliferate extensively and differentiate into the various neuronal subtypes and supporting cells that comprise the CNS. As such, hNPCs have tremendous potential for disease modeling, drug screening, and regenerative medicine applications. However, the use hNPCs for the study and treatment of neurological diseases requires the development of defined, robust, and scalable methods for their expansion and neuronal differentiation. To that end a rational design process was used to develop a vitronectin-derived peptide (VDP)-based substrate to support the growth and neuronal differentiation of hNPCs in conventional two-dimensional (2-D) culture and large-scale microcarrier (MC)-based suspension culture. Compared to hNPCs cultured on ECMP-based substrates, hNPCs grown on VDP-coated surfaces displayed similar morphologies, growth rates, and high expression levels of hNPC multipotency markers. Furthermore, VDP surfaces supported the directed differentiation of hNPCs to neurons at similar levels to cells differentiated on ECMP substrates. Here it has been demonstrated that VDP is a robust growth and differentiation matrix, as demonstrated by its ability to support the expansions and neuronal differentiation of hNPCs derived from three hESC (H9, HUES9, and HSF4) and one hiPSC (RiPSC) cell lines. Finally, it has been shown that VDP allows for the expansion or neuronal differentiation of hNPCs to quantities (>1010) necessary for drug screening or regenerative medicine purposes. In the future, the use of VDP as a defined culture substrate will significantly advance the clinical application of hNPCs and their derivatives as it will enable the large-scale expansion and neuronal differentiation of hNPCs in quantities necessary for disease modeling, drug screening, and regenerative medicine applications.

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2016