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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 biology is an emerging field which melds genetics, molecular biology, network theory, and mathematical systems to understand, build, and predict gene network behavior. As an engineering discipline, developing a mathematical understanding of the genetic circuits being studied is of fundamental importance. In this dissertation, mathematical concepts for understanding, predicting, and controlling gene transcriptional networks are presented and applied to two synthetic gene network contexts. First, this engineering approach is used to improve the function of the guide ribonucleic acid (gRNA)-targeted, dCas9-regulated transcriptional cascades through analysis and targeted modification of the RNA transcript. In so doing, a fluorescent guide RNA (fgRNA) is developed to more clearly observe gRNA dynamics and aid design. It is shown that through careful optimization, RNA Polymerase II (Pol II) driven gRNA transcripts can be strong enough to exhibit measurable cascading behavior, previously only shown in RNA Polymerase III (Pol III) circuits. Second, inherent gene expression noise is used to achieve precise fractional differentiation of a population. Mathematical methods are employed to predict and understand the observed behavior, and metrics for analyzing and quantifying similar differentiation kinetics are presented. Through careful mathematical analysis and simulation, coupled with experimental data, two methods for achieving ratio control are presented, with the optimal schema for any application being dependent on the noisiness of the system under study. Together, these studies push the boundaries of gene network control, with potential applications in stem cell differentiation, therapeutics, and bio-production.
ContributorsMenn, David J (Author) / Wang, Xiao (Thesis advisor) / Kiani, Samira (Committee member) / Haynes, Karmella (Committee member) / Nielsen, David (Committee member) / Marshall, Pamela (Committee member) / Arizona State University (Publisher)
Created2018
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 paper considers what factors influence student interest, motivation, and continued engagement. Studies show anticipated extrinsic rewards for activity participation have been shown to reduce intrinsic value for that activity. This might suggest that grade point average (GPA) has a similar effect on academic interests. Further, when incentives such as scholarships, internships, and careers are GPA-oriented, students must adopt performance goals in courses to guarantee success. However, performance goals have not been shown to correlated with continued interest in a topic. Current literature proposes that student involvement in extracurricular activities, focused study groups, and mentored research are crucial to student success. Further, students may express either a fixed or growth mindset, which influences their approach to challenges and opportunities for growth. The purpose of this study was to collect individual cases of students' experiences in college. The interview method was chosen to collect complex information that could not be gathered from standard surveys. To accomplish this, questions were developed based on content areas related to education and motivation theory. The content areas included activities and meaning, motivation, vision, and personal development. The developed interview method relied on broad questions that would be followed by specific "probing" questions. We hypothesize that this would result in participant-led discussions and unique narratives from the participant. Initial findings suggest that some of the questions were effective in eliciting detailed responses, though results were dependent on the interviewer. From the interviews we find that students value their group involvements, leadership opportunities, and relationships with mentors, which parallels results found in other studies.
ContributorsAbrams, Sara (Author) / Hartwell, Lee (Thesis director) / Correa, Kevin (Committee member) / Department of Psychology (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
This paper explores how marginalist economics defines and inevitably constrains Victorian sensation fiction's content and composition. I argue that economic intuition implies that sensationalist heroes and antagonists, writers and readers all pursued a fundamental, "rational" aim: the attainment of pleasure. So although "sensationalism" took on connotations of moral impropriety in the Victorian age, sensation fiction primarily involves experiences of pain on the page that excite the reader's pleasure. As such, sensationalism as a whole can be seen as a conformist product, one which mirrors the effects of all commodities on the market, rather than as a rebellious one. Indeed, contrary to modern and contemporary critics' assumptions, sensation fiction may not be as scandalous as it seems.
ContributorsFischer, Brett Andrew (Author) / Bivona, Daniel (Thesis director) / Looser, Devoney (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Economics Program in CLAS (Contributor) / School of Politics and Global Studies (Contributor) / Department of English (Contributor)
Created2014-12
Description
Through collection of survey data on the characteristics of college debaters, disparities in participation and success for women and racial and ethnic minorities are measured. This study then uses econometric tools to assess whether there is an in-group judging bias in college debate that systematically disadvantages female and minority participants. Debate is used as a testing ground for competing economic theories of taste-based and statistical discrimination, applied to a higher education context. The study finds persistent disparities in participation and success for female participants. Judges are more likely to vote for debaters who share their gender. There is also a significant disparity in the participation of racial and ethnic minority debaters and judges, as well as female judges.
ContributorsVered, Michelle Nicole (Author) / Silverman, Daniel (Thesis director) / Symonds, Adam (Committee member) / Dillon, Eleanor (Committee member) / Barrett, The Honors College (Contributor) / Economics Program in CLAS (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / School of Politics and Global Studies (Contributor)
Created2014-12
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
According to the Tax Policy Center, a joint project of the Brookings Institution and Urban Institute, the Earned Income Tax Credit (EITC) will provide 26 million households with 60 billion dollars of reduced taxes and refunds in 2015 \u2014 resources that serve to lift millions of families above the federal poverty line. Responding to the popularity of EITC programs and recent discussion of its expansion for childless adults, I select three comparative case studies of state-level EITC reform from 2005 to 2013. Each state represents a different kind of policy reform: the creation of a supplemental credit in Connecticut, credit reduction in New Jersey, and finally credit expansion for childless adults in Maryland. For each case study, I use Current Population Survey panel data from the March Supplement to complete a differences-in-differences (DD) analysis of EITC policy changes. Specifically, I analyze effects of policy reform on total earned income, employment and usual hours worked. For comparison groups, I construct unique counterfactual populations of northeastern U.S. states, using people of color with less than a college degree as my treatment group for their increased sensitivity to EITC policy reform. I find no statistically significant effects of policy creation in Connecticut, significant decreases in employment and hours worked in New Jersey, and finally, significant increases in earnings and hours worked in Maryland. My work supports the findings of other empirical work, suggesting that awareness of new supplemental EITC programs is critical to their effectiveness while demonstrating that these types of programs can affect the labor supply and outcomes of eligible groups.
ContributorsRichard, Katherine Rose (Author) / Dillon, Eleanor Wiske (Thesis director) / Silverman, Daniel (Committee member) / Herbst, Chris (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Economics Program in CLAS (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
A thorough understanding of the key concepts of logic is critical for student success. Logic is often not explicitly taught as its own subject in modern curriculums, which results in misconceptions among students as to what comprises logical reasoning. In addition, current standardized testing schemes often promote teaching styles which emphasize students' abilities to memorize set problem-solving methods over their capacities to reason abstractly and creatively. These phenomena, in tandem with halting progress in United States education compared to other developed nations, suggest that implementing logic courses into public schools and universities can better prepare students for professional careers and beyond. In particular, logic is essential for mathematics students as they transition from calculation-based courses to theoretical, proof-based classes. Many students find this adjustment difficult, and existing university-level courses which emphasize the technical aspects of symbolic logic do not fully bridge the gap between these two different approaches to mathematics. As a step towards resolving this problem, this project proposes a logic course which integrates historical, technical, and interdisciplinary investigations to present logic as a robust and meaningful subject warranting independent study. This course is designed with mathematics students in mind, with particular stresses on different formulations of deductively valid proof schemes. Additionally, this class can either be taught before existing logic classes in an effort to gradually expose students to logic over an extended period of time, or it can replace current logic courses as a more holistic introduction to the subject. The first section of the course investigates historical developments in studies of argumentation and logic throughout different civilizations; specifically, the works of ancient China, ancient India, ancient Greece, medieval Europe, and modernity are investigated. Along the way, several important themes are highlighted within appropriate historical contexts; these are often presented in an ad hoc way in courses emphasizing technical features of symbolic logic. After the motivations for modern symbolic logic are established, the key technical features of symbolic logic are presented, including: logical connectives, truth tables, logical equivalence, derivations, predicates, and quantifiers. Potential obstacles in students' understandings of these ideas are anticipated, and resolution methods are proposed. Finally, examples of how ideas of symbolic logic are manifested in many modern disciplines are presented. In particular, key concepts in game theory, computer science, biology, grammar, and mathematics are reformulated in the context of symbolic logic. By combining the three perspectives of historical context, technical aspects, and practical applications of symbolic logic, this course will ideally make logic a more meaningful and accessible subject for students.
ContributorsRyba, Austin (Author) / Vaz, Paul (Thesis director) / Jones, Donald (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Historical, Philosophical and Religious Studies (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05