Matching Items (15)
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- All Subjects: Synthetic Biology
- All Subjects: Learning
- Creators: Harrington Bioengineering Program
- Member of: Theses and Dissertations
- Resource Type: Text
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
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
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
Description
A previous study demonstrated that learning to lift an object is context-based and that in the presence of both the memory and visual cues, the acquired sensorimotor memory to manipulate an object in one context interferes with the performance of the same task in presence of visual information about a different context (Fu et al, 2012).
The purpose of this study is to know whether the primary motor cortex (M1) plays a role in the sensorimotor memory. It was hypothesized that temporary disruption of the M1 following the learning to minimize a tilt using a ‘L’ shaped object would negatively affect the retention of sensorimotor memory and thus reduce interference between the memory acquired in one context and the visual cues to perform the same task in a different context.
Significant findings were shown in blocks 1, 2, and 4. In block 3, subjects displayed insignificant amount of learning. However, it cannot be concluded that there is full interference in block 3. Therefore, looked into 3 effects in statistical analysis: the main effects of the blocks, the main effects of the trials, and the effects of the blocks and trials combined. From the block effects, there is a p-value of 0.001, and from the trial effects, the p-value is less than 0.001. Both of these effects indicate that there is learning occurring. However, when looking at the blocks * trials effects, we see a p-value of 0.002 < 0.05 indicating significant interaction between sensorimotor memories. Based on the results that were found, there is a presence of interference in all the blocks but not enough to justify the use of TMS in order to reduce interference because there is a partial reduction of interference from the control experiment. It is evident that the time delay might be the issue between context switches. By reducing the time delay between block 2 and 3 from 10 minutes to 5 minutes, I will hope to see significant learning to occur from the first trial to the second trial.
The purpose of this study is to know whether the primary motor cortex (M1) plays a role in the sensorimotor memory. It was hypothesized that temporary disruption of the M1 following the learning to minimize a tilt using a ‘L’ shaped object would negatively affect the retention of sensorimotor memory and thus reduce interference between the memory acquired in one context and the visual cues to perform the same task in a different context.
Significant findings were shown in blocks 1, 2, and 4. In block 3, subjects displayed insignificant amount of learning. However, it cannot be concluded that there is full interference in block 3. Therefore, looked into 3 effects in statistical analysis: the main effects of the blocks, the main effects of the trials, and the effects of the blocks and trials combined. From the block effects, there is a p-value of 0.001, and from the trial effects, the p-value is less than 0.001. Both of these effects indicate that there is learning occurring. However, when looking at the blocks * trials effects, we see a p-value of 0.002 < 0.05 indicating significant interaction between sensorimotor memories. Based on the results that were found, there is a presence of interference in all the blocks but not enough to justify the use of TMS in order to reduce interference because there is a partial reduction of interference from the control experiment. It is evident that the time delay might be the issue between context switches. By reducing the time delay between block 2 and 3 from 10 minutes to 5 minutes, I will hope to see significant learning to occur from the first trial to the second trial.
ContributorsHasan, Salman Bashir (Author) / Santello, Marco (Thesis director) / Kleim, Jeffrey (Committee member) / Helms Tillery, Stephen (Committee member) / Barrett, The Honors College (Contributor) / W. P. Carey School of Business (Contributor) / Harrington Bioengineering Program (Contributor)
Created2014-05
Description
Brain-computer interface technology establishes communication between the brain and a computer, allowing users to control devices, machines, or virtual objects using their thoughts. This study investigates optimal conditions to facilitate learning to operate this interface. It compares two biofeedback methods, which dictate the relationship between brain activity and the movement of a virtual ball in a target-hitting task. Preliminary results indicate that a method in which the position of the virtual object directly relates to the amplitude of brain signals is most conducive to success. In addition, this research explores learning in the context of neural signals during training with a BCI task. Specifically, it investigates whether subjects can adapt to parameters of the interface without guidance. This experiment prompts subjects to modulate brain signals spectrally, spatially, and temporally, as well differentially to discriminate between two different targets. However, subjects are not given knowledge regarding these desired changes, nor are they given instruction on how to move the virtual ball. Preliminary analysis of signal trends suggests that some successful participants are able to adapt brain wave activity in certain pre-specified locations and frequency bands over time in order to achieve control. Future studies will further explore these phenomena, and future BCI projects will be advised by these methods, which will give insight into the creation of more intuitive and reliable BCI technology.
ContributorsLancaster, Jenessa Mae (Co-author) / Appavu, Brian (Co-author) / Wahnoun, Remy (Co-author, Committee member) / Helms Tillery, Stephen (Thesis director) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor) / Department of Psychology (Contributor)
Created2014-05
Description
This paper explores the use of different classroom management styles by teachers engaged in a study. The study was focused on testing an educational computer program called The Doctor's Cure in s southwester school district with ready access to computers. The Doctor's Cure uses interactive storytelling and transformational play to teach seventh graders how to write persuasively. The definitions of student centered and teacher centered management styles used in this paper are drawn from Garret (2008) which suggests that teachers are not entirely one management style or the other, but a mix of the two. This paper closely examines three teachers, two with teacher centered styles and one with a student centered style in order to see which style was most effective in promoting the learning of persuasive writing skills. The findings tentatively indicate that teacher centered management styles yield larger gains in learning compared to more student centered styles.
ContributorsAyala, Joel Nicholas (Author) / Hayes, Elisabeth (Thesis director) / Siyahhan, Sinem (Committee member) / Holmes, Jeff (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2013-05
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 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
Older adults tend to learn at a lesser extent and slower rate than younger individuals. This is especially problematic for older adults at risk to injury or neurological disease who require therapy to learn and relearn motor skills. There is evidence that the reticulospinal system is critical to motor learning and that deficits in the reticulospinal system may be responsible, at least in part, for learning deficits in older adults. Specifically, delays in the reticulospinal system (measured via the startle reflex) are related to poor motor learning and retention in older adults. However, the mechanism underlying these delays in the reticulospinal system is currently unknown.
Along with aging, sleep deprivation is correlated with learning deficits. Research has shown that a lack of sleep negatively impacts motor skill learning and consolidation. Since there is a link between sleep and learning, as well as learning and the reticulospinal system, these observations raise the question: does sleep deprivation underlie reticulospinal delays? We hypothesized that sleep deprivation was correlated to a slower startle response, indicating a delayed reticulospinal system. Our objectives were to observe the impact of sleep deprivation on 1) the startle response (characterized by muscle onset latency and percentage of startle responses elicited) and 2) functional performance (to determine whether subjects were sufficiently sleep deprived).
21 young adults participated in two experimental sessions: one control session (8-10 hour time in bed opportunity for at least 3 nights prior) and one sleep deprivation session (0 hour time in bed opportunity for one night prior). The same protocol was conducted during each session. First, subjects were randomly exposed to 15 loud, startling acoustic stimuli of 120 dB. Electromyography (EMG) data measured muscle activity from the left and right sternocleidomastoid (LSCM and RSCM), biceps brachii, and triceps brachii. To assess functional performance, cognitive, balance, and motor tests were also administered. The EMG data were analyzed in MATLAB. A generalized linear mixed model was performed on LSCM and RSCM onset latencies. Paired t-tests were performed on the percentage of startle responses elicited and functional performance metrics. A p-value of less than 0.05 indicated significance.
Thirteen out of 21 participants displayed at least one startle response during their control and sleep deprived sessions and were further analyzed. No differences were found in onset latency (RSCM: control = 75.87 ± 21.94ms, sleep deprived = 82.06 ± 27.47ms; LSCM: control = 79.53 ± 17.85ms, sleep deprived = 78.48 ± 20.75ms) and percentage of startle responses elicited (control = 84.10 ± 15.53%; sleep deprived = 83.59 ± 18.58%) between the two sessions. However, significant differences were observed in reaction time, TUG with Dual time, and average balance time with the right leg up. Our data did not support our hypothesis; no significant differences were seen between subjects’ startle responses during the control and sleep deprived sessions. However, sleep deprivation was indicated with declines were observed in functional performance. Therefore, we concluded that sleep deprivation may not affect the startle response and underlie delays in the reticulospinal system.
Along with aging, sleep deprivation is correlated with learning deficits. Research has shown that a lack of sleep negatively impacts motor skill learning and consolidation. Since there is a link between sleep and learning, as well as learning and the reticulospinal system, these observations raise the question: does sleep deprivation underlie reticulospinal delays? We hypothesized that sleep deprivation was correlated to a slower startle response, indicating a delayed reticulospinal system. Our objectives were to observe the impact of sleep deprivation on 1) the startle response (characterized by muscle onset latency and percentage of startle responses elicited) and 2) functional performance (to determine whether subjects were sufficiently sleep deprived).
21 young adults participated in two experimental sessions: one control session (8-10 hour time in bed opportunity for at least 3 nights prior) and one sleep deprivation session (0 hour time in bed opportunity for one night prior). The same protocol was conducted during each session. First, subjects were randomly exposed to 15 loud, startling acoustic stimuli of 120 dB. Electromyography (EMG) data measured muscle activity from the left and right sternocleidomastoid (LSCM and RSCM), biceps brachii, and triceps brachii. To assess functional performance, cognitive, balance, and motor tests were also administered. The EMG data were analyzed in MATLAB. A generalized linear mixed model was performed on LSCM and RSCM onset latencies. Paired t-tests were performed on the percentage of startle responses elicited and functional performance metrics. A p-value of less than 0.05 indicated significance.
Thirteen out of 21 participants displayed at least one startle response during their control and sleep deprived sessions and were further analyzed. No differences were found in onset latency (RSCM: control = 75.87 ± 21.94ms, sleep deprived = 82.06 ± 27.47ms; LSCM: control = 79.53 ± 17.85ms, sleep deprived = 78.48 ± 20.75ms) and percentage of startle responses elicited (control = 84.10 ± 15.53%; sleep deprived = 83.59 ± 18.58%) between the two sessions. However, significant differences were observed in reaction time, TUG with Dual time, and average balance time with the right leg up. Our data did not support our hypothesis; no significant differences were seen between subjects’ startle responses during the control and sleep deprived sessions. However, sleep deprivation was indicated with declines were observed in functional performance. Therefore, we concluded that sleep deprivation may not affect the startle response and underlie delays in the reticulospinal system.
ContributorsGopalakrishnan, Smita (Author) / Honeycutt, Claire (Thesis director) / Petrov, Megan (Committee member) / Harrington Bioengineering Program (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Motor skill acquisition, the process by which individuals practice and consolidate
movement to become faster, more accurate and efficient, declines with age. Initial skill acquisition is dominated by cortical structures; however as learning proceeds, literature from
rodents and songbirds suggests that there is a transition away from cortical execution. Recent
evidence indicates that the reticulospinal system plays an important role in integration and
retention of learned motor skills. The brainstem has known age-rated deficits including cell
shrinkage & death. Given the role of the reticulospinal system in skill acquisition and older
adult’s poor capacity to learn, it begs the question: are delays in the reticulospinal system
associated with older adult’s poor capacity to learn?
Our objective was to evaluate if delays in the reticulospinal system (measured via the
startle reflex) and corticospinal system (measured via Transcranial Magnetic Stimulation (TMS) are correlated to impairment of motor learning in older adults. We found that individuals with fast startle responses resembling those of younger adults show the most improvement and retention while individuals with delayed startle responses show the least. We also found that there was no relationship between MEP latencies and improvement and retention. Moreover, linear regression analysis indicated that startle onset latency exists within a continuum of learning outcomes suggesting that startle onset latency may be a sensitive measure to predict learning deficits in older adults. As there exists no method to determine an individual’s relative learning capacity, these results open the possibility of startle, which is an easy and inexpensive behavioral measure and can be used to determine learning deficits in older adults to facilitate better dosing during rehabilitation therapy.
movement to become faster, more accurate and efficient, declines with age. Initial skill acquisition is dominated by cortical structures; however as learning proceeds, literature from
rodents and songbirds suggests that there is a transition away from cortical execution. Recent
evidence indicates that the reticulospinal system plays an important role in integration and
retention of learned motor skills. The brainstem has known age-rated deficits including cell
shrinkage & death. Given the role of the reticulospinal system in skill acquisition and older
adult’s poor capacity to learn, it begs the question: are delays in the reticulospinal system
associated with older adult’s poor capacity to learn?
Our objective was to evaluate if delays in the reticulospinal system (measured via the
startle reflex) and corticospinal system (measured via Transcranial Magnetic Stimulation (TMS) are correlated to impairment of motor learning in older adults. We found that individuals with fast startle responses resembling those of younger adults show the most improvement and retention while individuals with delayed startle responses show the least. We also found that there was no relationship between MEP latencies and improvement and retention. Moreover, linear regression analysis indicated that startle onset latency exists within a continuum of learning outcomes suggesting that startle onset latency may be a sensitive measure to predict learning deficits in older adults. As there exists no method to determine an individual’s relative learning capacity, these results open the possibility of startle, which is an easy and inexpensive behavioral measure and can be used to determine learning deficits in older adults to facilitate better dosing during rehabilitation therapy.
ContributorsRangarajan, Vishvak (Author) / Honeycutt, Claire (Thesis director) / Schaefer, Sydney (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Motor skill acquisition, the process by which individuals practice and consolidate movement to become faster, more accurate and efficient, declines with age. Initial skill acquisition is dominated by cortical structures; however as learning proceeds, literature from rodents and songbirds suggests that there is a transition away from cortical execution. Recent evidence indicates that the reticulospinal system plays an important role in integration and retention of learned motor skills. The brainstem has known age-rated deficits including cell shrinkage & death. Given the role of the reticulospinal system in skill acquisition and older adult’s poor capacity to learn, it begs the question: are delays in the reticulospinal system associated with older adult’s poor capacity to learn?
Our objective was to evaluate if delays in the reticulospinal system (measured via the startle reflex) are correlated to impairment of motor learning in older adults. We found that individuals with fast startle responses resembling those of younger adults show the most learning and retention of that learning while individuals with delayed startle responses show the least. Moreover, linear regression analysis indicated that startle onset latency exists within a continuum of learning outcomes suggesting that startle onset latency may be a sensitive measure to predict learning deficits in older adults. As there exists no method to determine an individual’s relative learning capacity, these results open the possibility of startle, which is an easy and inexpensive behavioral measure, being used to predict learning deficits in older adults to facilitate better dosing during rehabilitation therapy.
Our objective was to evaluate if delays in the reticulospinal system (measured via the startle reflex) are correlated to impairment of motor learning in older adults. We found that individuals with fast startle responses resembling those of younger adults show the most learning and retention of that learning while individuals with delayed startle responses show the least. Moreover, linear regression analysis indicated that startle onset latency exists within a continuum of learning outcomes suggesting that startle onset latency may be a sensitive measure to predict learning deficits in older adults. As there exists no method to determine an individual’s relative learning capacity, these results open the possibility of startle, which is an easy and inexpensive behavioral measure, being used to predict learning deficits in older adults to facilitate better dosing during rehabilitation therapy.
ContributorsSchreiber, Joseph James (Author) / Honeycutt, Claire (Thesis director) / Schaefer, Sydney (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05