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Description
Alzheimer’s disease is a major problem affecting over 5.7 million Americans. Although much is known about the effects of this neurogenerative disease, the exact pathogenesis is still unknown. One very important characteristic of Alzheimer’s is the accumulation of beta amyloid protein which often results in plaques. To understand these beta amyloid proteins better, antibody fragments may be used to bind to these oligomers and potentially reduce the effects of Alzheimer’s disease.
This thesis focused on the expression and crystallization the fragment antigen binding antibody fragment A4. A fragment antigen binding fragment was chosen to be worked with as it is more stable than many other antibody fragments. A4 is important in Alzheimer’s disease as it is able to identify toxic beta amyloid.
This thesis focused on the expression and crystallization the fragment antigen binding antibody fragment A4. A fragment antigen binding fragment was chosen to be worked with as it is more stable than many other antibody fragments. A4 is important in Alzheimer’s disease as it is able to identify toxic beta amyloid.
ContributorsColasurd, Paige (Author) / Nannenga, Brent (Thesis advisor) / Mills, Jeremy (Committee member) / Varman, Arul (Committee member) / Arizona State University (Publisher)
Created2018
Description
Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an obstacle. While chromatin provides another tool with which to control gene expression, regional density can lead to variability in genome editing efficiency by CRISPR/Cas9 systems. Many groups have attempted to de-silence chromatin to regulate genes and enhance DNA's accessibility to nucleases, but inconsistent results leave outstanding questions. Here, I test different types of activators, to analyze changes in chromatin features that result for chromatin opening, and to identify the critical biochemical features that support artificially generated open, transcriptionally active chromatin.
I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions.
SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug.
Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells.
I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions.
SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug.
Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells.
ContributorsBarrett, Cassandra M (Author) / Haynes, Karmella A (Thesis advisor) / Rege, Kaushal (Committee member) / Mills, Jeremy (Committee member) / Kiani, Samira (Committee member) / Arizona State University (Publisher)
Created2019
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
Turbidity is a known problem for UV water treatment systems as suspended particles can shield contaminants from the UV radiation. UV systems that utilize a reflective radiation chamber may be able to decrease the impact of turbidity on the efficacy of the system. The purpose of this study was to determine how kaolin clay and gram flour turbidity affects inactivation of Escherichia coli (E. coli) when using a UV system with a reflective chamber. Both sources of turbidity were shown to reduce the inactivation of E. coli with increasing concentrations. Overall, it was shown that increasing kaolin clay turbidity had a consistent effect on reducing UV inactivation across UV doses. Log inactivation was reduced by 1.48 log for the low UV dose and it was reduced by at least 1.31 log for the low UV dose. Gram flour had a similar effect to the clay at the lower UV dose, reducing log inactivation by 1.58 log. At the high UV dose, there was no change in UV inactivation with an increase in turbidity. In conclusion, turbidity has a significant impact on the efficacy of UV disinfection. Therefore, removing turbidity from water is an essential process to enhance UV efficiency for the disinfection of microbial pathogens.
ContributorsMalladi, Rohith (Author) / Abbaszadegan, Morteza (Thesis director) / Alum, Absar (Committee member) / Fox, Peter (Committee member) / School of Human Evolution & Social Change (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Accurately predicting local ranges of isotopic signatures in human populations is essential for answering questions about past migrations and mobility. While local ranges of δ18O can be estimated using modern baseline samples and precipitation models, there are many environmental and anthropogenic drivers that can cause these ranges to deviate from the ranges seen in human populations. This study performs a geostatistical meta-analysis on a large dataset (n = 1,370) of spatially contextualized archaeological δ18O samples from 30 publications in order to generate a predictive model of local human δ18O ranges in the Central Andes. Two models were generated, one using archaeological samples of both humans and fauna, and the other using only humans. The model using only human samples makes more accurate predictions, cautioning against the incorporation of faunal δ18O samples in studies of human provenance. The models are also compared against a model of δ18O values found in precipitation across the study area, and significant differences lead to the conclusion that precipitation models are insufficient for predicting local human δ18O ranges.
ContributorsHatley, Camden Miller (Author) / Knudson, Kelly (Thesis director) / Scaffidi, Beth (Committee member) / School of Earth and Space Exploration (Contributor) / School of Human Evolution & Social Change (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
This study evaluates medical pluralism among 1.5 generation Indian American immigrants. 1.5 generation Indian Americans (N=16) were surveyed regarding their engagement in complementary and alternative medical systems (CAM), how immigration affected that, and reasons for and for not continuing the use of CAM. Results indicated most 1.5 Indian immigrants currently engage in CAM, given that their parents also engage in CAM. The top reasons respondents indicated continued engagement in CAM was that it has no side effects and is preventative. Reasons for not practicing CAM included feeling out of place, not living with parents or not believing in CAM. After immigration, most participants decreased or stopped their engagement in CAM. More women than men continued to practice CAM after immigration. From the results, it was concluded that CAM is still important to 1.5 generation Indian immigrants.
ContributorsMurugesh, Subhiksha (Author) / Stotts, Rhian (Thesis director) / Mubayi, Anuj (Committee member) / School of Human Evolution & Social Change (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Forced pregnancy has been and remains a tactic of implementing genocide and inflicting long-lasting damage on a population. Forced marriages during the Cambodian genocide (1975-1979) and rape camps established during the Bosnian genocide (1992-1995) are two of many ways in which forced pregnancies can be implemented. This comparative study has identified social constructs within Bosnian and Cambodian cultures that allowed forced pregnancy to impact these populations. In the context of the Cambodian genocide, the Khmer Rouge implemented forced marriages in order to reproduce an agricultural labor force that would sustain the state of Democratic Kampuchea without foreign aid. The cultural construct of marriage promoted childbearing and sustained these marriages even after the fall of the Khmer Rouge regime. The Bosnian genocide, on the other hand, was an ethnic cleansing against Bosnian Muslims. Serbian forces established rape camps to impregnate Bosnian Muslim women in order to stigmatize them to the extent that they would (culturally) no longer be able to bear children for their own ethnic community. The cultural constructs of virginity and patrilineal descent acted as key factors in the effectiveness of forced pregnancy as a method of ethnic cleansing. While Bosnian Muslim rape camp survivors faced stigma for having been raped and for keeping their children if they chose to, Cambodian survivors would not. Cambodian women faced social expectations to stay in their marriages and keep their children in order to fulfill their duties as wives and mothers. In Bosnia, however, no social construct existed to support children born outside of marriage. In addition to these cultural constructs, various other factors influenced survivors' attitudes towards their children, including the presence of third party rapists in the Cambodian genocide and the fact that many Bosnian Muslim survivors did not know the identity of the father of their children. Comparative analysis of these two genocides has contributed to a more holistic understanding of the impacts of genocide and has informed how forced pregnancy operates across multiple cultural ideologies and lifestyles.
ContributorsVirdee, Tajinder (Author) / Holman, Christine (Thesis director) / Toth, Stephen (Committee member) / School of Human Evolution & Social Change (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
Description
The purpose of this research study was to examine the intersection of the relationship between homelessness and mental illness, including other factors such as substance abuse. A secondary purpose of this study was to gain an awareness of service delivery models and associated funding streams for providing services to homeless persons with mental illness. A thorough literature review was conducted by the author in order to aid in answering these questions. The author also conducted interviews with 27 homeless and formerly homeless clients living in Denver who were receiving services through the Colorado Coalition for the Homeless. Finally, the author conducted 4 qualitative interviews with policy experts who worked extensively in homeless services and advocacy in the Metro-Denver area. All data was entered into an Excel workbook, and a series of graphs and tables were made to present the research results. The themes of mental illness and substance abuse were common amongst the sample population, but the most common theme was that of the lack of affordable housing available. The majority of respondents also cited involvement in the criminal justice system such as incarceration, as well as family issues as major factors in them becoming homeless. The policy experts all cited the Housing First as well as the Permanent Supportive Housing model as the most effective service delivery model for those who are both homeless and mentally-ill, and Denver is utilizing some very innovative funding streams for these service delivery models. In conclusion, the author found through both the literature review and quantitative research, that homelessness is not truly a mental illness or substance abuse issue alone, though this relationship does hold clinical importance. Homelessness is instead the result of an excessive shortage of permanent and affordable housing units across the United States.
ContributorsNelson, Hunter Lee (Author) / Shafer, Michael (Thesis director) / Ferguson-Colvin, Kristin (Committee member) / School of Community Resources and Development (Contributor) / School of Human Evolution & Social Change (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
Description
Every minute and a half, an American is sexually assaulted (Department of Justice, 2017). After an instance of sexual assault, some victims are given the choice of having a sexual assault evidence kit (SAK) collected. These kits are designed to collect DNA evidence that will, in the best case scenario, result in the identification of the perpetrator. If the perpetrator cannot be located, the DNA profile can still be submitted to the FBI’s CODIS databank, which houses hundreds of thousands of DNA profiles from criminal cases, and may still lead to apprehension of the rapist. Unfortunately, some SAKs experience long delays, decades even, before being tested. To date, there are hundreds of thousands of untested SAKs that remain in police custody awaiting to be submitted for forensic profiling across the country. Here, we completed a holistic investigation of sexual assault response and SAK processing in Arizona. It is important to notice that the focus of our study not only includes SAK processing and the backlog but sexual assault prevention and improving victim reporting in an effort to understand the SAK “pipeline,” from assault to prosecution.
We identified problems in three major categories that negatively impact the SAK pipeline: historical inertia, legislative and institutional limitations, and community awareness. We found that a large number of SAKs in Arizona have remained untested due insufficient funding and staffing for public crime labs making it difficult for state labs to alleviate the SAK backlog while simultaneously responding to incoming cases (“Why the Backlog Exists,” n.d.). However, surveys of ASU undergraduate students revealed a significant interest in campus assault and the SAK backlog. Based on our findings, we suggest harnessing the interest of undergraduate students and recruiting them to specialized SAK-oriented forensic technician and sexual assault nurse examiner (SANE) training at ASU with the goal of creating a workforce that will alleviate the absence of trained professionals within the country. We also explore the possibility of the creation of a private crime laboratory at ASU devoted the processing of SAKs in Arizona as a measure of alleviating the demand on local public laboratories and providing a more economic alternative to commercial laboratories. The creation of an SAK laboratory at ASU would provide undergraduates the opportunity to learn more about real forensic analysis on campus, provide a pipeline for students to become technicians themselves, and help reduce and prevent a future SAK backlog in Arizona.
We identified problems in three major categories that negatively impact the SAK pipeline: historical inertia, legislative and institutional limitations, and community awareness. We found that a large number of SAKs in Arizona have remained untested due insufficient funding and staffing for public crime labs making it difficult for state labs to alleviate the SAK backlog while simultaneously responding to incoming cases (“Why the Backlog Exists,” n.d.). However, surveys of ASU undergraduate students revealed a significant interest in campus assault and the SAK backlog. Based on our findings, we suggest harnessing the interest of undergraduate students and recruiting them to specialized SAK-oriented forensic technician and sexual assault nurse examiner (SANE) training at ASU with the goal of creating a workforce that will alleviate the absence of trained professionals within the country. We also explore the possibility of the creation of a private crime laboratory at ASU devoted the processing of SAKs in Arizona as a measure of alleviating the demand on local public laboratories and providing a more economic alternative to commercial laboratories. The creation of an SAK laboratory at ASU would provide undergraduates the opportunity to learn more about real forensic analysis on campus, provide a pipeline for students to become technicians themselves, and help reduce and prevent a future SAK backlog in Arizona.
ContributorsStewart, Jamie (Co-author) / Brokaw, Danielle (Co-author) / Stone, Megan (Co-author) / Kanthaswamy, Sreetharan (Thesis director) / Oldt, Robert (Committee member) / School of Human Evolution & Social Change (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Growing hotspots of unvaccinated children corroborate with states that have highly permissive vaccination policies. State-based nonmedical exemption (NME) policies such as religious or philosophical exemptions make it easy for parents to opt out of vaccinating their children thus lowering herd immunity and increasing the risk of outbreaks. Recent studies have revealed that Phoenix is the metropolitan area with the highest number of nonmedical exemptions in the country with vaccination rates below herd immunity. This thesis investigates the role of the law in enabling low vaccination rates and develops a set of policy recommendations that lawmakers may use as a tool to restore these rates to herd immunity levels. To do this, an in-depth literature review was performed and supplemented with a case study of California’s policy response to the 2014 Disneyland measles outbreak. This information was synthesized into an in-depth policy analysis addressing the political, social, practical, and economic factors of the issue and four potential policy responses for state lawmakers. Based on the analysis and California’s example, eliminating nonmedical vaccine exemptions was identified as the most effective policy option to reach the intended goal of restoring vaccination rates to herd immunity levels. This policy option is both the most cost-effective and productive in reaching herd immunity but infringes the most on parental rights and will be met with the most significant political pushback. Despite these challenges, lawmakers should take this policy step to protect our community and the most vulnerable among us.
ContributorsSaxon, Mary Elizabeth (Author) / Reddy, Swapna (Thesis director) / Speer, Matthew (Committee member) / School of Human Evolution & Social Change (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05