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- Creators: Barrett, The Honors College
- 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
Current and recent trends in Galician sociolinguistics merit a study into the ongoing theoretical deadlock surrounding the standardization and normalization of Galego—the endonym of the local language. To this effect, this research aims primarily to i) evaluate the ideological frameworks of the two prevailing theories regarding the institutionalization of a Standard Galician (reintegrationism and isolationism) and the enduring conflict therein, ii) to cross-examine these theories' foundational assumptions and assertions with actual linguistic attitudes among the Galician (and, where relevant, Portuguese) public and their corresponding trends, and iii) to present relevant suggestions for practical mediation between the competing linguistic institutions advocating their own linguistic tenets with rigid austerity. By drawing upon previous academic material investigating these and other cultural, social, political and economic influences on the ongoing reestablishment of the Galician language, we can adequately appraise the transitional presence in modern Galician society as both a tool of communication and a symbol of sociocultural identity within Galicia and across its borders.
ContributorsDoyle-Matta, Joaquin (Author) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Abstract:
Moldova is one of the smallest and poorest countries in Europe, yet it has a rich and multifaceted history that makes it an interesting case study when exploring the complex connections between language and identity. In this paper, I aim to explore the situation of the Soviet Union and the identity of Moldovans in regards to language policies, as well as the historic and social landscapes that allowed such policies to take hold, such has Moldova's historical roots with Romania. Although it has been over twenty years since the fall of the Soviet Union, an iron curtain still deeply divides the country of Moldova, and the cleavages are felt and defined in terms of linguistic identity. While the true conflicts and issues at stake in Moldova run much deeper than differences in linguistic identity and in issues surrounding minority language rights (for example, economic concerns), such discord is consistently framed in terms of linguistic conflict—a central theme in the Republic of Moldova ever since Soviet influence began in the 1920s.
Moldova is one of the smallest and poorest countries in Europe, yet it has a rich and multifaceted history that makes it an interesting case study when exploring the complex connections between language and identity. In this paper, I aim to explore the situation of the Soviet Union and the identity of Moldovans in regards to language policies, as well as the historic and social landscapes that allowed such policies to take hold, such has Moldova's historical roots with Romania. Although it has been over twenty years since the fall of the Soviet Union, an iron curtain still deeply divides the country of Moldova, and the cleavages are felt and defined in terms of linguistic identity. While the true conflicts and issues at stake in Moldova run much deeper than differences in linguistic identity and in issues surrounding minority language rights (for example, economic concerns), such discord is consistently framed in terms of linguistic conflict—a central theme in the Republic of Moldova ever since Soviet influence began in the 1920s.
ContributorsForee, Emily (Author) / Bahtchevanova, Mariana (Committee member) / Danton, Naomi (Contributor) / Barrett, The Honors College (Contributor)
Created2014-05
Description
This paper investigates the usage of the Japanese sentence-final construction no(da) in 27 postings collected from online question-and-answer forums. This linguistic feature appears frequently in both spoken and written Japanese discourse; however, it continues to pose difficulties to researchers when attempting to identify its meaning and functions. A major issue is that insufficient information regarding discourse context has resulted in ambiguity in many of the functions described in previous studies. Considering these limitations, the present study attempted to specify discourse contexts towards which three proposed functions of no(da) gravitate: 1) giving and asking explanation, 2) evoking politeness, and 3) organizing discourse. The results of this study indicate that two collocations, ~no desu ga and ~no deshou ka, play important roles in serving these functions in the genre of question-and-answer forums.
ContributorsAlvillar, Satomi Noelle (Author) / Tanno, Koji (Thesis director) / Shimomura, Tomoko (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor)
Created2014-12
Description
As my year abroad in France was vastly approaching, it became apparent that the accessibility of certain resources would prove beneficial in the pursuit of my honors thesis in French. Thus, even before my departure it was decided that I would focus on the French texting language. While the specifics were not yet fully developed, it was certain that the subject was relevant as the use of communication technologies were becoming more prevalent amongst younger generations. Upon my arrival in Lyon, the objective of my thesis was realized as I began to slowly understand the descriptive nuances of both spoken French and the ever-changing texting language. As a language student, it felt necessary to understand and analyze both the prescriptive and descriptive facets of the French language, including those of the text shorthand. Therefore, it was my intention to learn the vocabulary and linguistic traits that were most frequented in this distinct jargon, not solely for myself, but also for my fellow students of French. This task was accomplished by reading and recording the text messages from ten native speakers. These texts were then compiled into a proper corpus, which was primarily used as the resource for the further applied projects I wished to create. Given this research, a type of dictionary was organized from the words that I found to be most exemplary of the French texting language. In addition, an analysis was also written concerning the common linguistics traits found in the corpus. It was my intent to provide the students of Arizona State University with relevant resources that could assist in the immersion process during or before their study abroad experience. In pursuit of any language, it seems that it is integral to maintain a sense of understanding of all common elements of a language, as they are constantly evolving. Thus, it was important for me in my attempt to master French that I understood all the nuances of the vernacular that were being used by the native speakers.
ContributorsMarquez, Monique Evangeline (Author) / Bahtchevanova, Mariana (Thesis director) / Danton, Naomi (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor)
Created2014-12
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
Bacteria have been shown to possess a large array of regulatory mechanisms to not just respond to a diverse array of environmental stresses, but to injurious artificial proteins as well. A previous investigation introduced DX, a man-made ATP sequestering protein into Escherichia coli (E. coli) which resulted in the formation of novel endoliposome structures and induced a viable but non-culturable state (VBNC) that was not easily reversed. It was hypothesized that the broadly conserved bacterial stringent response pathway may have been responsible for the observed phenotypic changes. With the goal of unveiling the molecular mechanism behind this novel response, changes in cellular morphology and physiology upon DX expression were assessed in a population of E. coli encoding a dysfunctional relA gene, one of the two genes controlling the induction of the stringent response. It was ultimately shown that RelA directly contributed to cellular filamentation, endoliposome structure formation, and the induction of a VBNC state. While the stringent response has been extensively shown to induce a VBNC state, to our knowledge, relA has not yet been shown to induce filamentation or coordinate the formation of endoliposome structures in bacteria. As the stringent response has been shown to be increasingly involved in antibiotic tolerance, this study provided an exciting opportunity to further characterize this adaptive response pathway to aid in the future development of novel therapeutics. In addition to this, this study continued to highlight that the DX protein may serve one of the first tools to allow for the direct selection of bacteria in a VBNC state by morphologically distinguishing non-culturable cells through cellular filamentation.
ContributorsFrost, Fredrick Charles (Author) / Chaput, John (Thesis director) / Wachter, Rebekka (Committee member) / Korch, Shaleen (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-12
Description
With an increasing global interest in Chinese economics and society, more and more native English speakers have started to learn Chinese as a second language (L2). While English and Chinese share a similar word order at the syntactic level, they differ significantly in ways to keep track of reference at the discourse level. There are generally three ways to keep track of reference: repeating the full noun phrase (NP), replacing the full NP with a lexical pronoun, or omitting the NP using zero anaphora. Chinese, a topic-prominent language, allows a wide use of zero anaphora to maintain reference; whereas English, a subject-prominent language, allows only a limited use of zero anaphora. Due to this difference, Chinese as a second language learners whose native language is English (CSL learners), must learn to implement the use of zero anaphora more frequently in their Chinese discourse. The purpose of this study is to investigate how CSL learners keep track of reference using zero anaphora. It is hypothesized that CSL learners at intermediate proficiency level would display a transfer effect from English to Chinese in their Chinese discourse. Specifically, they would produce fewer zero anaphora than native Chinese speakers, and they would also tend to consider discourse with many uses of zero anaphora for reference tracking as less appropriate. To test the hypothesis, a story-retelling task and multiple-choice questions task were adopted. The results of both tasks supported the hypothesis. Meanwhile, it is also evident that the CSL learners have acquired some usage of zero anaphora in their Chinese discourse as the usage of zero anaphora was more frequent when speaking Chinese than English.
ContributorsTang, Tianyu (Co-author) / Abdulla, Farzana (Co-author) / Chien, Jasmine (Co-author) / Zhang, Xia (Thesis director) / Bahtchevanova, Mariana (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of International Letters and Cultures (Contributor) / School of Human Evolution and Social Change (Contributor) / Department of Supply Chain Management (Contributor)
Created2015-05