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Over 5.8 million people are currently living with Alzheimer’s disease (AD), with the sixth highest mortality rate in the United States. No known cure or substantially life-extending treatment exists. With the growing aging population these numbers are only expected to increase to about 13.8 million by the year 2050. Alzheimer’s is a multifactorial disease, giving rise to two main types: familial AD (FAD) and sporadic AD (SAD). Although there are different factors associated with each type of the disease, both FAD and SAD result in neuronal and synaptic loss and remain difficult to model in-vitro and treat overall.
Current advances in cellular models of neurodegenerative diseases overcome a variety of limitations possessed in animal and post-mortem human models. Human-induced pluripotent stem cells (hiPSCs) provide a platform with cells that can self-renew and differentiate into mature and functional cell types. HiPSCs are at the forefront of neurodegenerative disease research because of their ability to differentiate into neural cell types. Apolipoprotein E (ApoE) is a protein encoded by the APOE gene found on chromosome 19 of the human genome. There are three common polymorphisms in the APOE gene, resulting from a single amino acid change in the protein. The presence of these polymorphisms are studied as associated risk factors of developing AD. Different combinations of these alleles closely relate to the risk a patient has in developing Alzheimer’s disease. The risk associated effects of this gene are primarily investigated, however the protective effects are not examined to the same extent.
This research aims to overcome the existing limitations in cell differentiations and improve cell population purity that limits the variables present in the culture. To do this, this study optimized a differentiation protocol by separating and purifying neuronal cell populations to study the potential protective effects associated with ApoE, a risk factor seen in SAD. This platform aims to use a purified cell population to effectively analyze cell type specific affects of the ApoE risk factor, specifically in neurons.
Current advances in cellular models of neurodegenerative diseases overcome a variety of limitations possessed in animal and post-mortem human models. Human-induced pluripotent stem cells (hiPSCs) provide a platform with cells that can self-renew and differentiate into mature and functional cell types. HiPSCs are at the forefront of neurodegenerative disease research because of their ability to differentiate into neural cell types. Apolipoprotein E (ApoE) is a protein encoded by the APOE gene found on chromosome 19 of the human genome. There are three common polymorphisms in the APOE gene, resulting from a single amino acid change in the protein. The presence of these polymorphisms are studied as associated risk factors of developing AD. Different combinations of these alleles closely relate to the risk a patient has in developing Alzheimer’s disease. The risk associated effects of this gene are primarily investigated, however the protective effects are not examined to the same extent.
This research aims to overcome the existing limitations in cell differentiations and improve cell population purity that limits the variables present in the culture. To do this, this study optimized a differentiation protocol by separating and purifying neuronal cell populations to study the potential protective effects associated with ApoE, a risk factor seen in SAD. This platform aims to use a purified cell population to effectively analyze cell type specific affects of the ApoE risk factor, specifically in neurons.
ContributorsFrisch, Carlye Arin (Author) / Brafman, David (Thesis director) / Tian, Xiaojun (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Effectively modeling Alzheimer’s disease will lend to a more comprehensive
understanding of the disease pathology, more efficacious drug development and
regenerative medicine as a form of treatment. There are limitations with current
transgenic mouse models of Alzheimer’s disease and the study of post mortem brain tissue of Alzheimer’s diseases patients. Stem cell models can overcome the lack of clinical relevance and impracticality associated with current models. Ideally, the use of stem cell models provides the foundation to study the biochemical and physiological aspects of Alzheimer’s disease, but at the cellular level. Moreover, the future of drug development and disease modeling can be improved by developing a reproducible and well-characterized model of AD that can be scaled up to meet requirements for basic and translational applications. Characterization and analysis of a heterogenic neuronal culture developed from induced pluripotent stem cells calls for the understanding of single cell identity and cell viability. A method to analyze RNA following intracellular sorting was developed in order to analyze single cell identity of a heterogenic population
of human induced pluripotent stem cells and neural progenitor cells. The population was intracellularly stained and sorted for Oct4. RNA was isolated and analyzed with qPCR, which demonstrated expected expression profiles for Oct4+ and Oct4- cells. In addition, a protocol to label cells with pO2 sensing nanoprobes was developed to assess cell viability. Non-destructive nanoprobe up-take by neural progenitor cells was assessed with fluorescent imaging and flow cytometry. Nanoprobe labeled neurons were cultured long-term and continued to fluoresce at day 28. The proof of concept experiments demonstrated will be further expanded upon and utilized in developing a more clinically relevant and cost-effective model of Alzheimer’s disease with downstream applications
in drug development and regenerative medicine.
understanding of the disease pathology, more efficacious drug development and
regenerative medicine as a form of treatment. There are limitations with current
transgenic mouse models of Alzheimer’s disease and the study of post mortem brain tissue of Alzheimer’s diseases patients. Stem cell models can overcome the lack of clinical relevance and impracticality associated with current models. Ideally, the use of stem cell models provides the foundation to study the biochemical and physiological aspects of Alzheimer’s disease, but at the cellular level. Moreover, the future of drug development and disease modeling can be improved by developing a reproducible and well-characterized model of AD that can be scaled up to meet requirements for basic and translational applications. Characterization and analysis of a heterogenic neuronal culture developed from induced pluripotent stem cells calls for the understanding of single cell identity and cell viability. A method to analyze RNA following intracellular sorting was developed in order to analyze single cell identity of a heterogenic population
of human induced pluripotent stem cells and neural progenitor cells. The population was intracellularly stained and sorted for Oct4. RNA was isolated and analyzed with qPCR, which demonstrated expected expression profiles for Oct4+ and Oct4- cells. In addition, a protocol to label cells with pO2 sensing nanoprobes was developed to assess cell viability. Non-destructive nanoprobe up-take by neural progenitor cells was assessed with fluorescent imaging and flow cytometry. Nanoprobe labeled neurons were cultured long-term and continued to fluoresce at day 28. The proof of concept experiments demonstrated will be further expanded upon and utilized in developing a more clinically relevant and cost-effective model of Alzheimer’s disease with downstream applications
in drug development and regenerative medicine.
ContributorsKnittel, Jacob James (Author) / Brafman, David (Thesis director) / Salvatore, Oddo (Committee member) / School of Life Sciences (Contributor) / Dean, W.P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Cell fate is a complex and dynamic process with many genetic components. It has often been likened to “multistable” mathematical systems because of the numerous possible “stable” states, or cell types, that cells may end up in. Due to its complexity, understanding the process of cell fate and differentiation has proven challenging. A better understanding of cell differentiation has applications in regenerative stem cell therapies, disease pathologies, and gene regulatory networks.
A variety of different genes have been associated with cell fate. For example, the Nanog/Oct-4/Sox2 network forms the core interaction of a gene network that maintains stem cell pluripotency, and Oct-4 and Sox2 also play a role in the tissue types that stem cells eventually differentiate into. Using the CRISPR/cas9 based homology independent targeted integration (HITI) method developed by Suzuki et al., we can integrate fluorescent tags behind genes with reasonable efficiency via the non-homologous end joining (NHEJ) DNA repair pathway. With human embryonic kidney (HEK) 293T cells, which can be transfected with high efficiencies, we aim to create a three-parameter reporter cell line with fluorescent tags for three different genes related to cell fate. This cell line would provide several advantages for the study of cell fate, including the ability to quantitatively measure cell state, observe expression heterogeneity among a population of genetically identical cells, and easily monitor fluctuations in expression patterns.
The project is partially complete at this time. This report discusses progress thus far, as well as the challenges faced and the future steps for completing the reporter line.
A variety of different genes have been associated with cell fate. For example, the Nanog/Oct-4/Sox2 network forms the core interaction of a gene network that maintains stem cell pluripotency, and Oct-4 and Sox2 also play a role in the tissue types that stem cells eventually differentiate into. Using the CRISPR/cas9 based homology independent targeted integration (HITI) method developed by Suzuki et al., we can integrate fluorescent tags behind genes with reasonable efficiency via the non-homologous end joining (NHEJ) DNA repair pathway. With human embryonic kidney (HEK) 293T cells, which can be transfected with high efficiencies, we aim to create a three-parameter reporter cell line with fluorescent tags for three different genes related to cell fate. This cell line would provide several advantages for the study of cell fate, including the ability to quantitatively measure cell state, observe expression heterogeneity among a population of genetically identical cells, and easily monitor fluctuations in expression patterns.
The project is partially complete at this time. This report discusses progress thus far, as well as the challenges faced and the future steps for completing the reporter line.
ContributorsLoveday, Tristan Andre (Author) / Wang, Xiao (Thesis director) / Brafman, David (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Chronic Traumatic Encephalopathy (CTE) is a neurodegenerative brain disease that results from repetitive brain trauma causing brain structure, personality, behavioral, and cognitive changes. CTE is currently undiagnosable and untreatable in living patients. This thesis investigates research surrounding CTE and presents a comparative discussion of the advantages and disadvantages of current diagnostic methods used for other neurodegenerative diseases that may be useful for the diagnosis of CTE.
ContributorsBlair, Sierra (Co-author) / Blair, Taylor (Co-author) / Brafman, David (Thesis director) / Stabenfeldt, Sarah (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The issue of wild horse management in the American West has become prevalent in the media recently and management strategies are often criticized and opposed by the public. Horses have been a core feature of American history and culture nearly since the colonization of the western frontier, and popular media such as television and movies paint a romantic but often unrealistic picture of wild horses. Land management agencies must balance limited resources with an ever-growing wild horse population in order to properly manage public land so that it retains its ecological integrity and is still able to be used by multiple stakeholders, and they also must endure public criticism throughout the process. I used a photo elicitation survey to gather responses to photographic images of wild horses and determine how the public feels about wild horse management, given that horses are seen as a symbol of freedom and the American West. It was revealed that people who are unfamiliar with the issue still have opinions about how the horses should be managed, and these opinions often mirror what can be found in popular media.
ContributorsDuran, Kiana Alexis (Author) / Minteer, Ben (Thesis director) / Schoon, Michael (Thesis director) / Murphree, Julie (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
As human populations continue to expand, interactions with wildlife are expected to increase due to destruction of land and global climate change threatening native habitats. Established areas of protection are becoming essential to species survival and biodiversity protection. National Parks (NP) are a globally ubiquitous method employed to protect wildlife and habitats. Often NPs are mosaics of relatively small protected areas in a “sea” of human-dominated landscapes, and these remaining habitat “islands” are becoming essential to preventing species extinction. However, the establishment of a NP can lead to increased human-wildlife conflicts (HWC) and disenfranchisement of local communities, particularly along their borders. We conducted semi-structured interviews in six different countries to better understand the nature of HWCs at the borders of major NPs: (1) Khao Yai NP, Thailand; (2) Myall Lakes NP, Australia; (3) Chitwan NP, Nepal; (4) Kruger NP, South Africa; (5) Chingaza NP, Colombia, and (6) Yellowstone NP, United States. We evaluated affinity to wildlife, perception of conflicts, management success, and potential solutions at each park to better understand the global nature of HWCs.We also evaluated these data in relationship to the Human Development Index (HDI) to determine if there was a correlation between development and conflict issues. We found the intrinsic value of wildlife to not markedly differ between countries. Conflict was perceived as higher in the United States and Australia but was known to be of greater intensity in Nepal and South Africa. Management of NPs was well-regarded with a slight decrease from less-developed countries to more-developed countries, with solutions that were creative and unique to each region. Results appeared to be related to shifting baselines between countries and also to equivalency in a cross-cultural assessment. When these theories are taken into account, the complexity of HWCs globally is better understood. As our world continues to expand and NPs become some of the only contiguous native habitat and refuges for wildlife, it is important to understand the complex relationships occurring at the interface between natural and human communities and to explore effective solutions to these problems.
ContributorsRagan, Kinley Ann (Author) / Schoon, Michael (Thesis director) / Schipper, Jan (Committee member) / Senko, Jesse (Committee member) / School of Life Sciences (Contributor, Contributor) / School of Geographical Sciences and Urban Planning (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Human activities around the world are threatening scores of wildlife species, pushing them closer to extinction. In order to address what many conservationists view as a global biodiversity crisis, it is vital that more people are inspired to care about wild animals and motivated to act in ways that help protect them. The up-close experiences and personal connections that people form with wild animals in zoos accredited by the Association of Zoos and Aquariums (AZA) or the World Association of Zoos and Aquariums (WAZA) can help achieve this. However, it is not very well understood how different types of encounters within these zoos may inspire conservation mindedness and pro-environmental behaviors. During this thesis project, surveys were conducted at the AZA-accredited Arizona Center for Nature Conservation/Phoenix Zoo to understand how interactive, hands-on animal experiences within zoos differ from passively viewing zoo animals when it comes to inspiring people to care about conservation. The Phoenix Zoo is home to two different species of giraffes, and guests can view them from the front of the Savanna Exhibit. Guests can also participate in the Giraffe Encounter, which is a much more interactive, hands-on experience. After surveying guests at both locations, the results showed that fewer people at the Giraffe Encounter responded that they often engage in pro-environmental behaviors. This may indicate that the people who participated in the Giraffe Encounter came to the zoo more for recreation and entertainment than to learn about wildlife. Despite this, more people learned something new about nature or conservation at the Giraffe Encounter than they did at the Savanna Exhibit. On average, guests also felt that the Giraffe Encounter motivated them to learn more about how to help animals in the wild than the Savanna Exhibit did. Overall, there is a strong correlation between having an interactive, hands-on experience with a zoo animal and caring more about wildlife conservation. However, more research still needs to be done in order to conclusively provide evidence for causation.
ContributorsBurgess, Christa Noell (Author) / Schoon, Michael (Thesis director) / Minteer, Ben (Committee member) / Allard, Ruth (Committee member) / School of Life Sciences (Contributor) / School of Sustainability (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-12
Description
This paper covers the wild horse overpopulation case study at the Salt River in Arizona, exploring how Traditional Ecological Knowledge (TEK) might help foster solutions to a lengthy and heated controversy about how to manage wild horses and burros on the rangeland. Fikret Berke's Sacred Ecology defines traditional ecological knowledge as, "a cumulative body of knowledge, practice, and belief evolving by adaptive processes and handed down through generations by cultural transmission, about the relationship of living beings (including humans) with one another and with their environment," (Berkes, 3). In contrast to current management strategies, TEK utilizes knowledge that comes from direct experience and intuitive knowing, rather than science-based, techno-rational streams of knowledge. Drawing on three modern sustainability concepts that support and stem from TEK, including: everything is connected, complex solutions can further complicate problems and diversity as a key to resilience, this paper sets forth a number of specific solutions to be considered moving forward, guided by the wisdom of TEK.
ContributorsLyford, Rebecca (Author) / Schoon, Michael (Thesis director) / Murphey, Julia (Committee member) / School of Sustainability (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Alzheimer’s Disease (AD) affects over 5 million individuals in the U.S. and has a direct cost estimated in excess of $200 billion per year. Broadly speaking, there are two forms of AD—early-onset, familial AD (FAD) and late-onset-sporadic AD (SAD). Animal models of AD, which rely on the overexpression of FAD-related mutations, have provided important insights into the disease. However, these models do not display important disease-related pathologies and have been limited in their ability to model the complex genetics associated with SAD.
Advances in cellular reprogramming, have enabled the generation of in vitro disease models that can be used to dissect disease mechanisms and evaluate potential therapeutics. To that end, efforts by many groups, including the Brafman laboratory, to generated patient-specific hiPSCs have demonstrated the promise of studying AD in a simplified and accessible system. However, neurons generated from these hiPSCs have shown some, but not all, of the early molecular and cellular hallmarks associated with the disease. Additionally, phenotypes and pathological hallmarks associated with later stages of the human disease have not been observed with current hiPSC-based systems. Further, disease relevant phenotypes in neurons generated from SAD hiPSCs have been highly variable or largely absent. Finally, the reprogramming process erases phenotypes associated with cellular aging and, as a result, iPSC-derived neurons more closely resemble fetal brain rather than adult brain.
It is well-established that in vivo cells reside within a complex 3-D microenvironment that plays a significant role in regulating cell behavior. Signaling and other cellular functions, such as gene expression and differentiation potential, differ in 3-D cultures compared with 2-D substrates. Nonetheless, previous studies using AD hiPSCs have relied on 2-D neuronal culture models that do not reflect the 3-D complexity of native brain tissue, and therefore, are unable to replicate all aspects of AD pathogenesis. Further, the reprogramming process erases cellular aging phenotypes. To address these limitations, this project aimed to develop bioengineering methods for the generation of 3-D organoid-based cultures that mimic in vivo cortical tissue, and to generate an inducible gene repression system to recapitulate cellular aging hallmarks.
Advances in cellular reprogramming, have enabled the generation of in vitro disease models that can be used to dissect disease mechanisms and evaluate potential therapeutics. To that end, efforts by many groups, including the Brafman laboratory, to generated patient-specific hiPSCs have demonstrated the promise of studying AD in a simplified and accessible system. However, neurons generated from these hiPSCs have shown some, but not all, of the early molecular and cellular hallmarks associated with the disease. Additionally, phenotypes and pathological hallmarks associated with later stages of the human disease have not been observed with current hiPSC-based systems. Further, disease relevant phenotypes in neurons generated from SAD hiPSCs have been highly variable or largely absent. Finally, the reprogramming process erases phenotypes associated with cellular aging and, as a result, iPSC-derived neurons more closely resemble fetal brain rather than adult brain.
It is well-established that in vivo cells reside within a complex 3-D microenvironment that plays a significant role in regulating cell behavior. Signaling and other cellular functions, such as gene expression and differentiation potential, differ in 3-D cultures compared with 2-D substrates. Nonetheless, previous studies using AD hiPSCs have relied on 2-D neuronal culture models that do not reflect the 3-D complexity of native brain tissue, and therefore, are unable to replicate all aspects of AD pathogenesis. Further, the reprogramming process erases cellular aging phenotypes. To address these limitations, this project aimed to develop bioengineering methods for the generation of 3-D organoid-based cultures that mimic in vivo cortical tissue, and to generate an inducible gene repression system to recapitulate cellular aging hallmarks.
ContributorsBounds, Lexi Rose (Author) / Brafman, David (Thesis director) / Wang, Xiao (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Circles of Sustainability is a self-evaluation tool designed to build educator capacity in K-12 schools seeking sustainability solutions. Based on the Sustainable Schools Challenge Handbook from Memphis, Tennessee, Circles of Sustainability considers environmental impact and efficiency, a healthy and safe school environment, sustainability and environmental education, and engagement and empowerment as four key pillars of whole-school sustainability. Each pillar is composed of elements and rubric items, which are reviewed, totaled, and colored in on the front page of the tool to help educators visualize and evaluate the current state of sustainability at their school. Since its first iteration completed in May 2017, the tool has been used by 300 educators throughout the United States during ASU's Sustainability Teachers' Academy (STA) workshops. Circles of Sustainability is completed as part of an activity called "Evaluating Your Community," where educators complete the tool and then brainstorm sustainability projects and solutions for their school and community. This paper is a review and discussion of the research, informal feedback and formal feedback used to create the second iteration of the tool. A second iteration of the tool was created to make the tool more user-friendly and ensure each pillar, element, and rubric item are based in research. The informal feedback was conducted during STA workshops in Tempe, Arizona; Abingdon, Virginia; Princeton, New Jersey; Chicago, Illinois; Los Angeles, California; Tucson, Arizona; and Charlotte, North Carolina. The formal feedback was conducted using a survey distributed to teachers who participated in the Tucson and Charlotte workshops. Overall, educators have responded positively to the tool, and the second iteration will continue to be used in future STA workshops throughout the United States.
ContributorsColbert, Julia (Author) / Schoon, Michael (Thesis director) / Merritt, Eileen (Committee member) / School of Sustainability (Contributor) / Division of Teacher Preparation (Contributor) / School of Geographical Sciences and Urban Planning (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05