Matching Items (28)
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- All Subjects: Traumatic Brain Injury
- All Subjects: Synthetic Biology
- Creators: Harrington Bioengineering Program
- Creators: Brafman, David
- Status: Published
Description
Currently in synthetic biology only the Las, Lux, and Rhl quorum sensing pathways have been adapted for broad engineering use. Quorum sensing allows a means of cell to cell communication in which a designated sender cell produces quorum sensing molecules that modify gene expression of a designated receiver cell. While useful, these three quorum sensing pathways exhibit a nontrivial level of crosstalk, hindering robust engineering and leading to unexpected effects in a given design. To address the lack of orthogonality among these three quorum sensing pathways, previous scientists have attempted to perform directed evolution on components of the quorum sensing pathway. While a powerful tool, directed evolution is limited by the subspace that is defined by the protein. For this reason, we take an evolutionary biology approach to identify new orthogonal quorum sensing networks and test these networks for cross-talk with currently-used networks. By charting characteristics of acyl homoserine lactone (AHL) molecules used across quorum sensing pathways in nature, we have identified favorable candidate pathways likely to display orthogonality. These include Aub, Bja, Bra, Cer, Esa, Las, Lux, Rhl, Rpa, and Sin, which we have begun constructing and testing. Our synthetic circuits express GFP in response to a quorum sensing molecule, allowing quantitative measurement of orthogonality between pairs. By determining orthogonal quorum sensing pairs, we hope to identify and adapt novel quorum sensing pathways for robust use in higher-order genetic circuits.
ContributorsMuller, Ryan (Author) / Haynes, Karmella (Thesis director) / Wang, Xiao (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
The purpose of this research was to determine and evaluate glutamate oxidase's ability to detect levels of glutamate as part of a working sensor capable of quantifying and detecting stress within the body in the case of adverse neurological events such as traumatic brain injury. Using electrochemical impedance spectroscopy (EIS), a linear dynamic range of glutamate was detected with a slope of 36.604 z/ohm/[pg/mL], a lower detection limit at 12.417 pg/mL, correlation of 0.97, and an optimal binding frequency of 117.20 Hz. After running through a frequency sweep the binding frequency was determined based on the highest consistent reproducibility and slope. The sensor was found to be specific against literature researched non-targets glucose, albumin, and epinephrine and working in dilutions of whole blood up to a concentration of 25%. With the implementation of Nafion, the sensor had a 250% improvement in signal and 155% improvement in correlation in 90% whole blood, illustrating the promise of a working blood sensor. Future work includes longitudinal studies and utilizing mesoporous carbon as the immobilization platform and incorporating this as part of a continuous, multiplexed blood sensor with glucose oxidase.
ContributorsLam, Alexandria Nicole (Author) / LaBelle, Jeffrey (Thesis director) / Ankeny, Casey (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
DescriptionMy main goal for my thesis is in conjunction with the research I started in the summer of 2010 regarding the creation of a TBI continuous-time sensor. Such goals include: characterizing the proteins in sensing targets while immobilized, while free in solution, and while in free solution in the blood.
ContributorsHaselwood, Brittney (Author) / LaBelle, Jeffrey (Thesis director) / Pizziconi, Vincent (Committee member) / Cook, Curtiss (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2011-12
Description
Electrochemical sensors function by detecting electroactive species at the electrode surface of a screen printed sensor. As more force is applied, the concentration of electroactive species at the surface of the sensor increases and a larger current is measured. Thus, when all conditions including voltage are made constant, as in Amp i-t, a quantifiable current can be read and the force applied can be calculated. Two common electrochemical techniques in which current is measured, cyclic voltammetry(CV) and amperometric i-t(Amp i-t), were used. A compressible sensor capable of transducing a force and acquiring feedback was created.
ContributorsFeldman, Austin Marc (Author) / LaBelle, Jeffrey (Thesis director) / Pizziconi, Vincent (Committee member) / Santello, Marco (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2013-05
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 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.
ContributorsRumbo, Kailey Michelle (Author) / Stabenfeldt, Sarah (Thesis director) / Kodibagkar, Vikram (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2014-05
Description
The endogenous response of neural stem cell/progenitor (NPSC) recruitment to the brain injury environment following a traumatic brain injury (TBI) is currently under heavy investigation. Mechanisms controlling NPSC proliferation and migration to the brain injury environment remain unclear; however, it is thought that the vascular extracellular matrix proteins (e.g. laminin, fibronectin, and vitronectin) and vascular endothelial growth factor (VEGF) play a role in mediating NPSC behavior through vasophillic interactions. This project attempts to uncover potential VEGF-ECM crosstalk in mediating migration and proliferation. To investigate migration, neurospheres were seeded on ECM-coated wells supplemented with VEGF and without VEGF, and neural outgrowth was measured at days 0, 1, 3, and 8 using differential interference contrast microscopy. Furthermore, single-cell NPSCs were seeded on ECM-coated Transwell membranes with VEGF supplemented media on one side and without VEGF to look at chemotactic migration. Migrated NPSCs were visualized with DAPI nuclear stain and imaged with an inverted fluorescent microscope. To investigate NPSC proliferation, NPSCs were seeded on ECM coated plates as in the radial migration assay and visualized with EdU on day 8. Total proliferation was measured by seeding NPSCs on ECM coated 96-well plates and incubating them with MTT on days 3 and 6. Proliferation was measured using a spectrophotometer at 630nm and 570nm wavelengths. It was found that VEGF-laminin crosstalk synergistically increased radial migration, but may not play a role in chemotactic migration. Understanding the mechanisms behind VEGF-laminin crosstalk in NPSC proliferation and migration may provide crucial information for the design of stem cell transplantation therapies in the future.
ContributorsMillar-Haskell, Catherine Susan (Author) / Stabenfeldt, Sarah (Thesis director) / Addington, Caroline (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2015-05
Description
Transgene expression in mammalian cells has been shown to meet resistance in the form of silencing due to chromatin buildup within the cell. Interactions of proteins with chromatin modulate gene expression profiles. Synthetic Polycomb transcription factor (PcTF) variants have the potential to reactivate these silence transgenes as shown in Haynes & Silver 2011. PcTF variants have been constructed via TypeIIS assembly to further investigate this ability to reactive transgenes. Expression in mammalian cells was confirmed via fluorescence microscopy and red fluorescent protein (RFP) expression in cell lysate. Examination of any variation in conferment of binding strength of homologous Polycomb chromodomains (PCDs) to its trimethylated lysine residue target on histone three (H3K27me3) was investigated using a thermal shift assay. Results indicate that PcTF may not be a suitable protein for surveying with SYPRO Orange, a dye that produces a detectable signal when exposed to the hydrophobic domains of the melting protein. A cell line with inducible silencing of a chemiluminescent protein was used to determine the effects PcTF variants had on gene reactivation. Results show down-regulation of the target reporter gene. We propose this may be due to PcTF not binding to its target; this would cause PcTF to deplete transcriptional machinery in the nucleus. Alternatively, the CMV promoter could be sequestering transcriptional machinery in its hyperactive transcription of PcTF leading to widespread down-regulation. Finally, the activation domain used may not be appropriate for this cell type. Future PcTF variants will address these hypotheses by including multiple Polycomb chromodomains (PCDs) to alter the binding dynamics of PcTF to its target, and by incorporating alternative promoters and activation domains.
ContributorsGardner, Cameron Lee (Author) / Haynes, Karmella (Thesis director) / Stabenfeldt, Sarah (Committee member) / Barrett, The Honors College (Contributor) / Department of Finance (Contributor) / Harrington Bioengineering Program (Contributor)
Created2015-05
Description
Traumatic brain injury (TBI) is a major cause of disability, with approximately 1.7 million incidents reported annually. Following a TBI, patients are likely to sustain sensorimotor and cognitive impairments and are at an increased risk of developing neurodegenerative diseases later in life. Despite this, robust therapies that treat TBI neuropathology are not available in the clinic. One emerging therapeutic approach is to target epigenetic mediators that modulate a variety of molecular regulatory events acutely following injury. Specifically, previous studies demonstrated that histone deacetylase inhibitor (HDACi) administration following TBI reduced inflammation, enhanced functional outcomes, and was neuroprotective. Here, we evaluated a novel quisinostat-loaded PLA-PEG nanoparticle (QNP) therapy in treating TBI as modeled by a controlled cortical impact. We evaluated initial pharmacodynamics within the injured cortex via histone acetylation levels following QNP treatment. We observed that QNP administration acutely following injury increased histone acetylation specifically within the injury penumbra, as detected by Western blot analysis. Given this effect, we evaluated QNP therapeutic efficacy. We observed that QNP treatment dampened motor deficits as measured by increased rotarod latency to fall relative to blank nanoparticle- and saline-treated controls. Additionally, open field results show that QNP treatment altered locomotion following injury. These results suggest that HDACi therapies are a beneficial therapeutic strategy following neural injury and demonstrate the utility for nanoparticle formulations as a mode for HDACi delivery following TBI.
ContributorsMousa, Gergey (Author) / Stabenfeldt, Sarah (Thesis director) / Newbern, Jason (Committee member) / Sirianni, Rachael (Committee member) / School of Life Sciences (Contributor) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Recent studies in traumatic brain injury (TBI) have found a temporal window where therapeutics on the nanometer scale can cross the blood-brain barrier and enter the parenchyma. Developing protein-based therapeutics is attractive for a number of reasons, yet, the production pipeline for high yield and consistent bioactive recombinant proteins remains a major obstacle. Previous studies for recombinant protein production has utilized gram-negative hosts such as Escherichia coli (E. coli) due to its well-established genetics and fast growth for recombinant protein production. However, using gram-negative hosts require lysis that calls for additional optimization and also introduces endotoxins and proteases that contribute to protein degradation. This project directly addressed this issue and evaluated the potential to use a gram-positive host such as Brevibacillus choshinensis (Brevi) which does not require lysis as the proteins are expressed directly into the supernatant. This host was utilized to produce variants of Stock 11 (S11) protein as a proof-of-concept towards this methodology. Variants of S11 were synthesized using different restriction enzymes which will alter the location of protein tags that may affect production or purification. Factors such as incubation time, incubation temperature, and media were optimized for each variant of S11 using a robust design of experiments. All variants of S11 were grown using optimized parameters prior to purification via affinity chromatography. Results showed the efficiency of using Brevi as a potential host for domain antibody production in the Stabenfeldt lab. Future aims will focus on troubleshooting the purification process to optimize the protein production pipeline.
ContributorsEmbrador, Glenna Bea Rebano (Author) / Stabenfeldt, Sarah (Thesis director) / Plaisier, Christopher (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Cell fate is a complex and dynamic process with many genetic components. It has often been likened to “multistable” mathematical systems because of the numerous possible “stable” states, or cell types, that cells may end up in. Due to its complexity, understanding the process of cell fate and differentiation has proven challenging. A better understanding of cell differentiation has applications in regenerative stem cell therapies, disease pathologies, and gene regulatory networks.
A variety of different genes have been associated with cell fate. For example, the Nanog/Oct-4/Sox2 network forms the core interaction of a gene network that maintains stem cell pluripotency, and Oct-4 and Sox2 also play a role in the tissue types that stem cells eventually differentiate into. Using the CRISPR/cas9 based homology independent targeted integration (HITI) method developed by Suzuki et al., we can integrate fluorescent tags behind genes with reasonable efficiency via the non-homologous end joining (NHEJ) DNA repair pathway. With human embryonic kidney (HEK) 293T cells, which can be transfected with high efficiencies, we aim to create a three-parameter reporter cell line with fluorescent tags for three different genes related to cell fate. This cell line would provide several advantages for the study of cell fate, including the ability to quantitatively measure cell state, observe expression heterogeneity among a population of genetically identical cells, and easily monitor fluctuations in expression patterns.
The project is partially complete at this time. This report discusses progress thus far, as well as the challenges faced and the future steps for completing the reporter line.
A variety of different genes have been associated with cell fate. For example, the Nanog/Oct-4/Sox2 network forms the core interaction of a gene network that maintains stem cell pluripotency, and Oct-4 and Sox2 also play a role in the tissue types that stem cells eventually differentiate into. Using the CRISPR/cas9 based homology independent targeted integration (HITI) method developed by Suzuki et al., we can integrate fluorescent tags behind genes with reasonable efficiency via the non-homologous end joining (NHEJ) DNA repair pathway. With human embryonic kidney (HEK) 293T cells, which can be transfected with high efficiencies, we aim to create a three-parameter reporter cell line with fluorescent tags for three different genes related to cell fate. This cell line would provide several advantages for the study of cell fate, including the ability to quantitatively measure cell state, observe expression heterogeneity among a population of genetically identical cells, and easily monitor fluctuations in expression patterns.
The project is partially complete at this time. This report discusses progress thus far, as well as the challenges faced and the future steps for completing the reporter line.
ContributorsLoveday, Tristan Andre (Author) / Wang, Xiao (Thesis director) / Brafman, David (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05