Matching Items (27)
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- All Subjects: Linguistics
- Creators: Department of English
- Status: Published
Description
The portability of genetic tools from one organism to another is a cornerstone of synthetic biology. The shared biological language of DNA-to-RNA-to-protein allows for expression of polypeptide chains in phylogenetically distant organisms with little modification. The tools and contexts are diverse, ranging from catalytic RNAs in cell-free systems to bacterial proteins expressed in human cell lines, yet they exhibit an organizing principle: that genes and proteins may be treated as modular units that can be moved from their native organism to a novel one. However, protein behavior is always unpredictable; drop-in functionality is not guaranteed.
My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli.
1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin.
2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists.
My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli.
1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin.
2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists.
ContributorsDaer, René (Author) / Haynes, Karmella (Thesis advisor) / Brafman, David (Committee member) / Nielsen, David (Committee member) / Kiani, Samira (Committee member) / Arizona State University (Publisher)
Created2017
Description
Synthetic manipulation of chromatin dynamics has applications for medicine, agriculture, and biotechnology. However, progress in this area requires the identification of design rules for engineering chromatin systems. In this thesis, I discuss research that has elucidated the intrinsic properties of histone binding proteins (HBP), and apply this knowledge to engineer novel chromatin binding effectors. Results from the experiments described herein demonstrate that the histone binding domain from chromobox protein homolog 8 (CBX8) is portable and can be customized to alter its endogenous function. First, I developed an assay to identify engineered fusion proteins that bind histone post translational modifications (PTMs) in vitro and regulate genes near the same histone PTMs in living cells. This assay will be useful for assaying the function of synthetic histone PTM-binding actuators and probes. Next, I investigated the activity of a novel, dual histone PTM binding domain regulator called Pc2TF. I characterized Pc2TF in vitro and in cells and show it has enhanced binding and transcriptional activation compared to a single binding domain fusion called Polycomb Transcription Factor (PcTF). These results indicate that valency can be used to tune the activity of synthetic histone-binding transcriptional regulators. Then, I report the delivery of PcTF fused to a cell penetrating peptide (CPP) TAT, called CP-PcTF. I treated 2D U-2 OS bone cancer cells with CP-PcTF, followed by RNA sequencing to identify genes regulated by CP-PcTF. I also showed that 3D spheroids treated with CP-PcTF show delayed growth. This preliminary work demonstrated that an epigenetic effector fused to a CPP can enable entry and regulation of genes in U-2 OS cells through DNA independent interactions. Finally, I described and validated a new screening method that combines the versatility of in vitro transcription and translation (IVTT) expressed protein coupled with the histone tail microarrays. Using Pc2TF as an example, I demonstrated that this assay is capable of determining binding and specificity of a synthetic HBP. I conclude by outlining future work toward engineering HBPs using techniques such as directed evolution and rational design. In conclusion, this work outlines a foundation to engineer and deliver synthetic chromatin effectors.
ContributorsTekel, Stefan (Author) / Haynes, Karmella (Thesis advisor) / Mills, Jeremy (Committee member) / Caplan, Michael (Committee member) / Brafman, David (Committee member) / Arizona State University (Publisher)
Created2019
Description
This is a constructed language made for fictional creatures that will be featured in a novel. The creatures are feral goblins, and they are based on Irish mythology. The novel that the goblins and the language will be featured in is titled MaddConn, and it was written by Jon Bendera. It is an urban fantasy novel. The goblins have a language in the novel because there are a number of goblin characters throughout the story. One of the main characters, Maddi, has to survive amongst the feral goblins for a portion of the novel while another main character, Connor, works his way to her. She has multiple close encounters with them, and also has to find her way around their territory, so she comes into contact with the language many times. Although the language in the story was created to have developed prior to humanity, this constructed language was made to look somewhat similar to human language. As it will be involved in fiction that will be read by humans, it needs to be easily understood by humans. The language reveals features of the goblin culture and anatomy. For instance, it can be seen in the language that their shallow, wide mouths and thin lips influence their phonology. Their physiology influences much of their language. Likewise, they have three fingers on each hand, which influences their number system. As the goblins and their language developed before humans and because they are not human creatures, the typology and word order do not follow all of the correlations found in human languages. All in all, the lexicon includes many words that have to do with three of their favorite things: violence, food, and work.
ContributorsMartinez, Randi Nicole (Author) / Van Gelderen, Elly (Thesis director) / Maring, Heather (Committee member) / Schreyer, Christine (Committee member) / Department of English (Contributor, Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
The rise of Italian in Sicily contrasts with a fierce regional pride that makes it difficult to determine the possible fate of Sicilian. This project focuses on a sociolinguistic analysis of the dialect of Sicilian spoken in and around Catania, Sicily. While there are programs in place to protect the language, the institutionalization of Italian in Sicily may be encroaching on Sicilian's use, especially with younger generations. The lure of the more industrialized North creates a culture of immigration in Sicily, which increasingly rewards the use of Italian. Using information from background research, a survey analyzing sociolinguistic factors and the individual's fluency in and use of Sicilian was created. The data from the survey showed that while understanding of Sicilian was fairly universal among participants, an individual's use and proficiency in Sicilian were most influenced by age and current place of residence (inside or outside Sicily). Younger people tended to know and use Sicilian less, and older participants tended to be more confident in their abilities and to use Sicilian more often. This is slightly complicated by an additional trend among participants currently living outside of Sicily towards a lower level of use and knowledge of Sicilian. All participants placed a significant emphasis on maintaining the ability to speak Sicilian, and on Sicilian language as an integral part of Sicilian culture.
ContributorsScaduto, Leah Christine (Author) / Dal Martello, Chiara (Thesis director) / Adams, Karen (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / School of Politics and Global Studies (Contributor) / Sandra Day O'Connor College of Law (Contributor) / Department of English (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
The goal of this thesis research was to figure out if there were tangible differences between the way men and women speak on Twitter, a micro blogging social media site, and to see if there ways to apply it to strategizing marketing campaigns. AntConc, a free concordance software by Lawrence Anthony, was used to help organize and analyze a corpus created from the Tweets that were collected form the public accounts of twelve different popular public figures. These individuals were chosen based on their profession or the industry that they are associated with, as well as their general popularity. The research focused on three main industries or professions that can be viewed as ‘gendered;’ which were ‘Modeling,’ ‘Fashion Publications,’ and ‘Sports.’ The data was then analyzed across five different main categories which included, ‘Additional Media,’ ‘Adjective Usage,’ ‘How are they talking?,’ ‘Who are they talking about?, and ‘What are they talking about?’ The primary data, along with secondary research was used to see if they words and language use of men and women aligned with stereotypical patterns or if there were patterns that were unique and overlooked.
What was found was that although gender did play a large part in the way men and women spoke, there were more similarities when comparing individuals of the same industry or profession, than there were if they were simply analyzed just based on gender. Additionally, there were many factors that made it difficult to say whether these were qualified patterns or simply tendencies. More research into this would be able to help marketing companies and individuals, better target the audience they want for social media campaigns, by taking into account the importance in contemporary differences in language use by men and women. However, this research would have to be done on data from sites like Twitter to provide an accurate depiction of the way men and women, on these very unique mediums, speak.
What was found was that although gender did play a large part in the way men and women spoke, there were more similarities when comparing individuals of the same industry or profession, than there were if they were simply analyzed just based on gender. Additionally, there were many factors that made it difficult to say whether these were qualified patterns or simply tendencies. More research into this would be able to help marketing companies and individuals, better target the audience they want for social media campaigns, by taking into account the importance in contemporary differences in language use by men and women. However, this research would have to be done on data from sites like Twitter to provide an accurate depiction of the way men and women, on these very unique mediums, speak.
ContributorsChan, Kayla Rose (Author) / Adams, Karen (Thesis director) / Shinabarger, Amy D. (Committee member) / Barrett, The Honors College (Contributor) / Department of Marketing (Contributor) / Department of English (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
Contributing to the small but growing body of research on linguistic discrimination in legal settings, this thesis conducts a sociolinguistic investigation of the impact of an individual's accent on juror perceptions of defendant favorability and innocence. The study used an online questionnaire in which sixty mock jurors were each asked to evaluate the audio testimony of a defendant representing one of three English ethnolects: African American, British South African, or Caucasian American. In addition to rating the defendant's persuasiveness, honesty, credibility, trustworthiness, and guilt, participants were also asked to determine an appropriate punishment (if any) for the defendant. Results indicate a preference of participants to issue an unsure or caveat opinion for the African American speaker but not to the British South African or Caucasian American speaker. The implications of these findings, as well as the correlations between each variable are discussed. The paper concludes with a recommendation for legal training and a revision of courtroom practices.
ContributorsMaerowitz, Max Robert (Author) / Prior, Matthew (Thesis director) / Adams, Karen (Committee member) / Barrett, The Honors College (Contributor) / School of Historical, Philosophical and Religious Studies (Contributor) / Department of English (Contributor)
Created2014-05
Description
This study observes two fanfiction speech communities, Danny Phantom and Detective Conan. The members of these communities write stories based upon the canon within these two animated cartoons and interact with one another through reviews, author's notes, and story summaries. Using the speech community model, this community's unique practices and communicative repertoire will be identified and analyzed. Both of these fandoms show similarities with the overarching general fanfiction speech community, but they also possess key differences that define them as their own separate community. Fan jargon is used frequently in author's notes, reviews, and summaries to indicate fan expertise and membership within the fandom as well as exclude newcomers from understanding the information. This jargon remains largely the same across languages, and using it properly is important to being considered a true fan. Furthermore, many stories share similar elements that are not present within the source material, indicating that the fandoms possess a shared communicative repertoire. Review practices also show strong cultural norms that demand that reviewers offer praise and encouragement to the writers. Most criticism is phrased extremely kindly to avoid breaking cultural norms. Those who do not follow these cultural norms are shunned by the community, and required to apologize to maintain proper fan membership. Fan hierarchy is also examined, including the ways that big name fans and reviewers exert centripetal and centrifugal forces upon the language, simultaneously pushing it towards standardization and variation. Authors also use many face saving techniques to demonstrate their own lack of knowledge within the community, especially if they are new or inexperienced. The members of these communities share a deep cultural connection that is strengthened by their practices and repertoires.
ContributorsDial, Ashtyn Nicole (Author) / Friedrich, Patricia (Thesis director) / O'Connor, Brendan (Committee member) / Computing and Informatics Program (Contributor) / Department of English (Contributor, Contributor, Contributor) / School of Music (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05