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- Creators: School of Mathematical and Statistical Sciences
- Creators: Mendez, Jose
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
The passage of 2007's Legal Arizona Workers Act, which required all new hires to be tested for legal employment status through the federal E-Verify database, drastically changed the employment prospects for undocumented workers in the state. Using data from the 2007-2010 American Community Survey, this paper seeks to identify the impact of this law on the labor force in Arizona, specifically regarding undocumented workers and less educated native workers. Overall, the data shows that the wage bias against undocumented immigrants doubled in the four years studied, and the wages of native workers without a high school degree saw a temporary, positive increase compared to comparable workers in other states. The law did not have an effect on the wages of native workers with a high school degree.
ContributorsSantiago, Maria Christina (Author) / Pereira, Claudiney (Thesis director) / Mendez, Jose (Committee member) / School of International Letters and Cultures (Contributor) / Department of Economics (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The January 12, 2010 Haiti earthquake, which hit Port-au-Prince in the late afternoon, was the cause of over 220,000 deaths and $8 billion in damages \u2014 roughly 120% of national GDP at the time. A Mw 7.5 earthquake struck rural Guatemala in the early morning in 1976 and caused 23,000-25,000 deaths, three times as many injuries, and roughly $1.1 billion in damages, which accounted for approximately 30% of Guatemala's GDP. The earthquake which hit just outside of Christchurch, New Zealand early in the morning on September 4, 2010 had a magnitude of 7.1 and caused just two injuries, no deaths, and roughly 7.2 billion USD in damages (5% of GDP). These three earthquakes, all with magnitudes over 7 on the Richter scale, caused extremely varied amounts of economic damage for these three countries. This thesis aims to identify a possible explanation as to why this was the case and suggest ways in which to improve disaster risk management going forward.
ContributorsHeuermann, Jamie Lynne (Author) / Schoellman, Todd (Thesis director) / Mendez, Jose (Committee member) / Department of Supply Chain Management (Contributor) / Department of Economics (Contributor) / W. P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
The following paper consists of a review of sovereign debt sustainability economics and IMF debt sustainability frameworks, as well as a historical case study of Greece and a variable suggestion for the IMF to improve baseline assumptions. The purpose of this paper is to review the current methodology of perceiving debt and improve upon it in the face of an increasingly indebted global economy. Thus, this paper suggests the IMF adopt the variable calculated in Reinhart and Rogoff (2009) as a new benchmark for determining debt sustainability of market access countries. Through an exploration of the most recent Greek crisis, as well as modern Greek financial and political history, the author of this paper contends the IMF should reduce the broadness of the MAC DSA, as it will make for better debt sustainability projections and assumptions in implementing debt program policy.
ContributorsJennings, Zane Phillips (Author) / Mendez, Jose (Thesis director) / Roberts, Nancy (Committee member) / Economics Program in CLAS (Contributor) / School of Politics and Global Studies (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
This project looks at the effects of American sanctions on the Sudanese economy. The purpose of the research is to evaluate the effects of the sanctions on the GDP (based on Purchasing Power Parity) of Sudan using linear regression analysis. We used a linear model to conduct analysis that included variables such as Sudan's trading partners, distance between Sudan and said partners, the GDP of these other countries, and whether there are sanctions imposed. The data collected runs from 1980 to 2011 \u2014 the year South Sudan became independent. The results of the analysis indicate that sanctions are ineffective in achieving their purpose which is ending the human rights violations in Sudan. The findings are consistent with arguments put forth by economics for decades.
ContributorsAli, Safa (Author) / Mendez, Jose (Thesis director) / Ali, Souad T. (Committee member) / Barrett, The Honors College (Contributor) / School of Criminology and Criminal Justice (Contributor) / Department of Economics (Contributor)
Created2014-12
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