Matching Items (12)
Filtering by

Clear all filters

136265-Thumbnail Image.png
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

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
136262-Thumbnail Image.png
Description
The Larynx plays a pivotal role in our ability to breathe and to speak. It is in our best interest to continue improving the status of tissue regeneration concerning the larynx so that patient voice quality of life can be less hindered in the face of laryngeal cancers and diseases.

The Larynx plays a pivotal role in our ability to breathe and to speak. It is in our best interest to continue improving the status of tissue regeneration concerning the larynx so that patient voice quality of life can be less hindered in the face of laryngeal cancers and diseases. Modern technology can allow us to use CT scans for both diagnosis and treatment. This medical imaging can be converted into three-dimensional patient specific models that are actualized through 3D printing. These implants improve upon the current state of the art because they can be produced in a timely manner, are developed with materials and methods ensuring their biocompatibility, and follow architectures and geometries best suited for the patient to improve their voice quality of life. Additionally they should be able to allow patient speech in the case of partial laryngectomies where the arytenoid has been removed by acting as a permanent vocal fold This treatment process for laryngectomies aligns itself with personalized medicine by targeting its geometry based on that of the patient. Technologies and manufacturing processes utilized to produce them are accessible and could all be used within the clinical space. The life-saving implant required for the laryngectomy healing and recovery process can be ready to implant for the patient within a few days of imaging them.
ContributorsBarry, Colin Patrick (Author) / Pizziconi, Vincent (Thesis director) / Lott, David (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2015-05
136133-Thumbnail Image.png
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

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
133856-Thumbnail Image.png
Description
Synthetic biology is an emerging engineering disciple, which designs and controls biological systems for creation of materials, biosensors, biocomputing, and much more. To better control and engineer these systems, modular genetic components which allow for highly specific and high dynamic range genetic regulation are necessary. Currently the field struggles to

Synthetic biology is an emerging engineering disciple, which designs and controls biological systems for creation of materials, biosensors, biocomputing, and much more. To better control and engineer these systems, modular genetic components which allow for highly specific and high dynamic range genetic regulation are necessary. Currently the field struggles to demonstrate reliable regulators which are programmable and specific, yet also allow for a high dynamic range of control. Inspired by the characteristics of the RNA toehold switch in E. coli, this project attempts utilize artificial introns and complementary trans-acting RNAs for gene regulation in a eukaryote host, S. cerevisiae. Following modification to an artificial intron, splicing control with RNA hairpins was demonstrated. Temperature shifts led to increased protein production likely due to increased splicing due to hairpin loosening. Progress is underway to demonstrate trans-acting RNA interaction to control splicing. With continued development, we hope to provide a programmable, specific, and effective means for translational gene regulation in S. cerevisae.
ContributorsDorr, Brandon Arthur (Author) / Wang, Xiao (Thesis director) / Green, Alexander (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
135297-Thumbnail Image.png
Description
Current research into live-cell dynamics, particularly those relating to chromatin structure and remodeling, are limited. The tools that are used to detect state changes in chromatin, such as Chromatin Immunoprecipitation and qPCR, require that the cell be killed off. This limits the ability of researchers to pinpoint changes in live

Current research into live-cell dynamics, particularly those relating to chromatin structure and remodeling, are limited. The tools that are used to detect state changes in chromatin, such as Chromatin Immunoprecipitation and qPCR, require that the cell be killed off. This limits the ability of researchers to pinpoint changes in live cells over a longer period of time. As such, there is a need for a live-cell sensor that can detect chromatin state changes. The Chromometer is a transgenic chromatin state sensor designed to better understand human cell fate and the chromatin changes that occur. HOXD11.12, a DNA sequence that attracts repressive Polycomb group (PCG) proteins, was placed upstream of a core promoter-driven fluorescent reporter (AmCyan fluorescent protein, CFP) to link chromatin repression to a CFP signal. The transgene was stably inserted at an ectopic site in U2-OS (osteosarcoma) cells. Expression of CFP should reflect the epigenetic state at the HOXD locus, where several genes are regulated by Polycomb to control cell differentiation. U2-OS cells were transfected with the transgene and grown under selective pressure. Twelve colonies were identified as having integrated parts from the transgene into their genomes. PCR testing verified 2 cell lines that contain the complete transgene. Flow cytometry indicated mono-modal and bimodal populations in all transgenic cell colonies. Further research must be done to determine the effectiveness of this device as a sensor for live cell state change detection.
ContributorsBarclay, David (Co-author) / Simper, Jan (Co-author) / Haynes, Karmella (Thesis director) / Brafman, David (Committee member) / School of Life Sciences (Contributor) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description

Pelvic organ prolapse (POP) is a condition involving the weakening of the pelvic floor, with a prevalence of up to 50% of women experiencing the condition to some degree. Individuals with the condition are susceptible to multiple symptoms include vaginal protrusion, dyspareunia, and difficulties with waste excretion. Risk factors are

Pelvic organ prolapse (POP) is a condition involving the weakening of the pelvic floor, with a prevalence of up to 50% of women experiencing the condition to some degree. Individuals with the condition are susceptible to multiple symptoms include vaginal protrusion, dyspareunia, and difficulties with waste excretion. Risk factors are common and numerous for POP, and the economic burden of the condition poses a significant cost to nations worldwide. For many years, the primary solution to POP was the usage of transvaginal meshes, often composed of polypropylene, but rising reports of harmful side effects have led to their recall. Due to this, the space is open for novel solutions, and treatments based in regenerative medicine are on the rise. One such potential treatment is the usage of functionalized polyvinyl alcohol scaffolds to support the regeneration and strengthening of the pelvic floor. To validate the usage of this scaffold, this study focuses on the biocompatibility of the scaffolds, with specific focus on the maintenance of cell viability and proliferation on the scaffold. Through usage of metabolic assays and fluorescence microscopy, scaffolds composed of functional polyvinyl alcohol with cellulose have shown promise in supporting the cell types necessary for reconstructing the pelvic floor.

ContributorsHaug, Joel (Author) / Song, Kenan (Thesis director) / Lancaster, Jessica (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor) / Harrington Bioengineering Program (Contributor)
Created2023-05
Description
Pelvic organ prolapse (POP) is a condition involving the weakening of the pelvic floor, with a prevalence of up to 50% of women experiencing the condition to some degree. Individuals with the condition are susceptible to multiple symptoms include vaginal protrusion, dyspareunia, and difficulties with waste excretion. Risk factors are

Pelvic organ prolapse (POP) is a condition involving the weakening of the pelvic floor, with a prevalence of up to 50% of women experiencing the condition to some degree. Individuals with the condition are susceptible to multiple symptoms include vaginal protrusion, dyspareunia, and difficulties with waste excretion. Risk factors are common and numerous for POP, and the economic burden of the condition poses a significant cost to nations worldwide. For many years, the primary solution to POP was the usage of transvaginal meshes, often composed of polypropylene, but rising reports of harmful side effects have led to their recall. Due to this, the space is open for novel solutions, and treatments based in regenerative medicine are on the rise. One such potential treatment is the usage of functionalized polyvinyl alcohol scaffolds to support the regeneration and strengthening of the pelvic floor. To validate the usage of this scaffold, this study focuses on the biocompatibility of the scaffolds, with specific focus on the maintenance of cell viability and proliferation on the scaffold. Through usage of metabolic assays and fluorescence microscopy, scaffolds composed of functional polyvinyl alcohol with cellulose have shown promise in supporting the cell types necessary for reconstructing the pelvic floor.
ContributorsHaug, Joel (Author) / Song, Kenan (Thesis director) / Lancaster, Jessica (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor) / Harrington Bioengineering Program (Contributor)
Created2023-05
Description
Pelvic organ prolapse (POP) is a condition involving the weakening of the pelvic floor, with a prevalence of up to 50% of women experiencing the condition to some degree. Individuals with the condition are susceptible to multiple symptoms include vaginal protrusion, dyspareunia, and difficulties with waste excretion. Risk factors are

Pelvic organ prolapse (POP) is a condition involving the weakening of the pelvic floor, with a prevalence of up to 50% of women experiencing the condition to some degree. Individuals with the condition are susceptible to multiple symptoms include vaginal protrusion, dyspareunia, and difficulties with waste excretion. Risk factors are common and numerous for POP, and the economic burden of the condition poses a significant cost to nations worldwide. For many years, the primary solution to POP was the usage of transvaginal meshes, often composed of polypropylene, but rising reports of harmful side effects have led to their recall. Due to this, the space is open for novel solutions, and treatments based in regenerative medicine are on the rise. One such potential treatment is the usage of functionalized polyvinyl alcohol scaffolds to support the regeneration and strengthening of the pelvic floor. To validate the usage of this scaffold, this study focuses on the biocompatibility of the scaffolds, with specific focus on the maintenance of cell viability and proliferation on the scaffold. Through usage of metabolic assays and fluorescence microscopy, scaffolds composed of functional polyvinyl alcohol with cellulose have shown promise in supporting the cell types necessary for reconstructing the pelvic floor.
ContributorsHaug, Joel (Author) / Song, Kenan (Thesis director) / Lancaster, Jessica (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor) / Harrington Bioengineering Program (Contributor)
Created2023-05
Description

Lab-grown food products of animal cell origin, now becoming popularly coined as, ‘Cellular Agriculture’ is a revolutionary breakthrough technology that has the potential to penetrate the lives of every American or citizen of the world. It is important to recognize that the impetus for developing this technology is fueled by

Lab-grown food products of animal cell origin, now becoming popularly coined as, ‘Cellular Agriculture’ is a revolutionary breakthrough technology that has the potential to penetrate the lives of every American or citizen of the world. It is important to recognize that the impetus for developing this technology is fueled by environmental concerns with climate change, rising geopolitical instability, and population growth projections, where farm-grown food has now become a growing national security issue. Notwithstanding its potential, in addition to the necessary technological innovation and economic scalability, the market success of cellular agriculture will depend greatly on regulatory oversight by multiple government agencies without which it can cause undue harm to individuals, populations, and the environment. Thus, it is critical for those appropriate United States governing bodies to ensure that the technology being developed is both safe and of an acceptable quality for human consumption and has no adverse environmental impact. As such, animal foods, derived from farms, previously regulated almost exclusively by the United States Department of Agriculture (USDA) are now being regulated under a joint formal agreement between the US Food and Drug Administration (US FDA) and the USDA if derived from the lab, i.e., lab-grown animal foods. The main reason for joint oversight between the FDA and the USDA is that the FDA has developed the in-house expertise to oversee primary cell harvesting and cell storage, as well as, cell growth and differentiation for the development of 3D-engineered tissues intended for tissue and organ replacement for the emerging field of regenerative medicine. As such, the FDA has been given the authority to oversee the ‘front end’ of lab-grown food processes which relies on the very same processes utilized in engineered human tissues to produce food-grade engineered tissues. Oversight then transitions to the USDA-FSIS (Food Safety and Inspection Service) during the harvesting stage of the cell culture process. The USDA-FSIS then oversees the further production and labeling of these products. Included in the agreement is the understanding that both bodies are responsible for communicating necessary information to each other and collaboratively developing new regulatory actions as needed. However, there currently lacks clarity on some topics regarding certain legal, ethical, and scientific issues. Lab-grown meat products require more extensive regulation than farm-grown animal food products to ensure that they are safe and nutritious for consumption. To do this, CFSAN can create new classes of lab-grown foods, such as ‘lab-grown USDA foods,’ ‘lab-grown non-USDA foods,’ ‘lab-grown extinct foods,’ ‘lab-grown human food tissues,’ and ‘medically activated lab-grown foods.’

ContributorsBanen, Samuel (Author) / Pizziconi, Vincent (Thesis director) / Feigal, David (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor) / School of Molecular Sciences (Contributor)
Created2023-05
132721-Thumbnail Image.png
Description
Tissue engineering is an emerging field focused on the repair, replacement, and regeneration of damaged tissue. Engineered tissue consists of three factors: cells, biomolecular signals, and a scaffold. Cell-free scaffolds present a unique opportunity to develop highly specific microenvironments with tunable properties. Norbornene-functionalized hyaluronic acid (NorHA) hydrogels provide spatial control

Tissue engineering is an emerging field focused on the repair, replacement, and regeneration of damaged tissue. Engineered tissue consists of three factors: cells, biomolecular signals, and a scaffold. Cell-free scaffolds present a unique opportunity to develop highly specific microenvironments with tunable properties. Norbornene-functionalized hyaluronic acid (NorHA) hydrogels provide spatial control over biomolecule binding through a photopolymerization process. With this, biomimetic gradients can be produced to model a variety of tissue interfaces. To produce these patterns, a gradient mechanism was developed to function in tandem with a syringe pump. A conversion equation was derived to calculate a panel speed from the volumetric flow rate setting on the pump. Seven speeds were used to produce fluorophore gradients on the surface of NorHA hydrogels to assess changes in the length and slope of the gradient. The results indicated a strong positive linear correlation between the speed of the panel and the length of the gradient as well as a strong negative correlation between the speed of the panel and the slope of the gradient. Additionally, the mechanism was able to successfully produce several other types of gradients including multiregional, dual, and triregional.
ContributorsSogge, Amber (Author) / Holloway, Julianne (Thesis director) / Stabenfeldt, Sarah (Committee member) / Fumasi, Fallon (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05