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Description
The GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the C9orf72 gene is the most common genetic abnormality associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastatingly progressive neurodegenerative diseases. The discovery of this genetic link confirmed that ALS and FTD reside along a spectrum with clinical and pathological commonalities. Historically understood as diseases resulting in neuronal death, the role of non-neuronal cells like astrocytes is still wholly unresolved. With evidence of cortical neurodegeneration leading to cognitive impairments in C9orf72-ALS/FTD, there is a need to investigate the role of cortical astrocytes in this disease spectrum. Here, a patient-derived induced pluripotent stem cell (iPSC) cortical astrocyte model was developed to investigate consequences of C9orf72-HRE pathogenic features in this cell type. Although there were no significant C9orf72-HRE pathogenic features in cortical astrocytes, transcriptomic, proteomic and phosphoproteomic profiles elucidated global disease-related phenotypes. Specifically, aberrant expression of astrocytic-synapse proteins and secreted factors were identified. SPARCL1, a pro-synaptogenic secreted astrocyte factor was found to be selectively decreased in C9orf72-ALS/FTD iPSC-cortical astrocytes. This finding was further validated in human tissue analyses, indicating that cortical astrocytes in C9orf72-ALS/FTD exhibit a reactive transformation that is characterized by a decrease in SPARCL1 expression. Considering the evidence for substantial astrogliosis and synaptic failure leading to cognitive impairments in C9orf72-ALS/FTD, these findings represent a novel understanding of how cortical astrocytes may contribute to the cortical neurodegeneration in this disease spectrum.
ContributorsBustos, Lynette (Author) / Sattler, Rita (Thesis advisor) / Newbern, Jason (Committee member) / Zarnescu, Daniela (Committee member) / Brafman, David (Committee member) / Mehta, Shwetal (Committee member) / Arizona State University (Publisher)
Created2023
Description
APOE encodes for a lipid transport protein and has three allelic variants-APOE ε2, ε3 and ε4 each of which differentially modulate the risk for Alzheimer’s disease (AD). The presence of the ε4 allele of APOE greatly increases AD risk compared to the presence of the more prevalent and risk neutral ε3 allele. An imbalance in the generation and clearance of amyloid beta (Aβ) peptides has been hypothesized to play a key role in driving the disease. APOE4 impacts several AD-relevant cellular processes. However, it is unclear whether these effects represent a gain of toxic function or a loss of function, specifically as it relates to modulating amyloid beta (Aβ) levels. Here, a set of APOE knockout (KO) and APOE4 isogenic human induced pluripotent stem cells (hiPSCs) were generated from a parental APOE3 hiPSC line with a highly penetrant familial AD (fAD) mutation to investigate this with respect to Aβ secretion in neural cultures and Aβ uptake in monocultures of microglia-like cells (iMGLs). Conversion of APOE3 to E4 as well as functionally knocking APOE out from the APOE3 parental line, result in elevated Aβ levels in neural cultures, likely through multiple mechanisms including the altered processing of the precursor protein to Aβ called amyloid precursor protein (APP). In pure neuronal cultures, a shift in the processing of APP was observed with the Aβ-generating amyloidogenic pathway being favored in both APOE3 as well as APOE4 neurons compared to APOE KO neurons, with APOE4 neurons exhibiting a greater shift. In iMGLs derived from the isogenic hiPSC lines, expression of APOE, regardless of the isoform, lowered the uptake of Aβ. Overall, APOE4 modulates Aβ levels through distinct loss of protective and gain of function effects. Dissecting these effects would contribute towards a better understanding of the design of potential APOE-targeted therapeutics in the future.
ContributorsRajaram Srinivasan, Gayathri (Author) / Brafman, David (Thesis advisor) / Plaisier, Christopher (Committee member) / Newbern, Jason (Committee member) / Stabenfeldt, Sarah (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2024
Description
Understanding the diversity, evolutionary relationships, and geographic distribution of species is foundational knowledge in biology. However, this knowledge is lacking for many diverse lineages of the tree of life. This is the case for the desert stink beetles in the tribe Amphidorini LeConte, 1862 (Coleoptera: Tenebrionidae) – a lineage of arid-adapted flightless beetles found throughout western North America. Four interconnected studies that jointly increase our knowledge of this group are presented. First, the darkling beetle fauna of the Algodones sand dunes in southern California is examined as a case study to explore the scientific practice of checklist creation. An updated list of the species known from this region is presented, with a critical focus on material now made available through digitization and global aggregation. This part concludes with recommendations for future biodiversity checklist authors. Second, the psammophilic genus Trogloderus LeConte, 1879 is revised. Six new species are described, and the first, multi-gene phylogeny for the genus is inferred. In addition, historical biogeographic reconstructions along with novel hypotheses of speciation patterns within the Intermountain Region are given. In particular, the Kaibab Plateau and Kaiparowitz Formation are found to have promoted speciation on the Colorado Plateau. The Owens Valley and prehistoric Bouse Embayment are similarly hypothesized to drive species diversification in southern California. Third, a novel phylogenomic analysis for the tribe Amphidorini is presented, based on 29 de novo partial transcriptomes. Three putative ortholog sets were discovered and analyzed to infer the relationships between species groups and genera. The existing classification of the tribe is found to be highly inadequate, though the earliest-diverging relationships within the tribe are still in question. Finally, the new phylogenetic framework is used to provide a genus-level revision for the Amphidorini, which previously contained six valid genera and 253 valid species. This updated classification includes more than 100 taxonomic changes and results in the revised tribe consisting of 16 genera, with three being described as new to science.
ContributorsJohnston, Murray Andrew (Author) / Franz, Nico M (Thesis advisor) / Cartwright, Reed (Committee member) / Taylor, Jesse (Committee member) / Pigg, Kathleen (Committee member) / Arizona State University (Publisher)
Created2018
Description
The ability to tolerate bouts of oxygen deprivation varies tremendously across the animal kingdom. Adult humans from different regions show large variation in tolerance to hypoxia; additionally, it is widely known that neonatal mammals are much more tolerant to anoxia than their adult counterparts, including in humans. Drosophila melanogaster are very anoxia-tolerant relative to mammals, with adults able to survive 12 h of anoxia, and represent a well-suited model for studying anoxia tolerance. Drosophila live in rotting, fermenting media and a result are more likely to experience environmental hypoxia; therefore, they could be expected to be more tolerant of anoxia than adults. However, adults have the capacity to survive anoxic exposure times ~8 times longer than larvae. This dissertation focuses on understanding the mechanisms responsible for variation in survival from anoxic exposure in the genetic model organism, Drosophila melanogaster, focused in particular on effects of developmental stage (larval vs. adults) and within-population variation among individuals.
Vertebrate studies suggest that surviving anoxia requires the maintenance of ATP despite the loss of aerobic metabolism in a manner that prevents a disruption of ionic homeostasis. Instead, the abilities to maintain a hypometabolic state with low ATP and tolerate large disturbances in ionic status appear to contribute to the higher anoxia tolerance of adults. Furthermore, metabolomics experiments support this notion by showing that larvae had higher metabolic rates during the initial 30 min of anoxia and that protective metabolites were upregulated in adults but not larvae. Lastly, I investigated the genetic variation in anoxia tolerance using a genome wide association study (GWAS) to identify target genes associated with anoxia tolerance. Results from the GWAS also suggest mechanisms related to protection from ionic and oxidative stress, in addition to a protective role for immune function.
Vertebrate studies suggest that surviving anoxia requires the maintenance of ATP despite the loss of aerobic metabolism in a manner that prevents a disruption of ionic homeostasis. Instead, the abilities to maintain a hypometabolic state with low ATP and tolerate large disturbances in ionic status appear to contribute to the higher anoxia tolerance of adults. Furthermore, metabolomics experiments support this notion by showing that larvae had higher metabolic rates during the initial 30 min of anoxia and that protective metabolites were upregulated in adults but not larvae. Lastly, I investigated the genetic variation in anoxia tolerance using a genome wide association study (GWAS) to identify target genes associated with anoxia tolerance. Results from the GWAS also suggest mechanisms related to protection from ionic and oxidative stress, in addition to a protective role for immune function.
ContributorsCampbell, Jacob B (Author) / Harrison, Jon F. (Thesis advisor) / Gadau, Juergen (Committee member) / Call, Gerald B (Committee member) / Sweazea, Karen L (Committee member) / Rosenberg, Michael S. (Committee member) / Arizona State University (Publisher)
Created2018
Description
Amongst the most studied of the social insects, the honey bee has a prominent place due to its economic importance and influence on human societies. Honey bee colonies can have over 50,000 individuals, whose activities are coordinated by chemical signals called pheromones. Because these pheromones are secreted from various exocrine glands, the proper development and function of these glands are vital to colony dynamics. In this thesis, I present a study of the developmental ontogeny of the exocrine glands found in the head of the honey bee. In Chapter 2, I elucidate how the larval salivary gland transitions to an adult salivary gland through apoptosis and cell growth, differentiation and migration. I also explain the development of the hypopharyngeal and the mandibular gland using apoptotic markers and cytoskeletal markers like tubulin and actin. I explain the fundamental developmental plan for the formation of the glands and show that apoptosis plays an important role in the transformation toward an adult gland.
ContributorsNath, Rachna (Author) / Gadau, Juergen (Thesis advisor) / Rawls, Alan (Committee member) / Harrison, Jon (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
Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, or amyotrophic lateral sclerosis are defined by the loss of several types of neurons and glial cells within the central nervous system (CNS). Combatting these diseases requires a robust population of relevant cell types that can be employed in cell therapies, drug screening, or patient specific disease modeling. Human induced pluripotent stem cells (hiPSC)-derived neural progenitor cells (hNPCs) have the ability to self-renew indefinitely and differentiate into the various neuronal and glial cell types of the CNS. In order to realize the potential of hNPCs, it is necessary to develop a xeno-free scalable platform for effective expansion and differentiation. Previous work in the Brafman lab led to the engineering of a chemically defined substrate—vitronectin derived peptide (VDP), which allows for the long-term expansion and differentiation of hNPCs. In this work, we use this substrate as the basis for a microcarrier (MC)-based suspension culture system. Several independently derived hNPC lines were cultured on MCs for multiple passages as well as efficiently differentiated to neurons. Finally, this MC-based system was used in conjunction with a low shear rotating wall vessel (RWV) bioreactor for the integrated, large-scale expansion and neuronal differentiation of hNPCs. Finally, VDP was shown to support the differentiation of hNPCs into functional astrocytes. Overall, this fully defined and scalable biomanufacturing system will facilitate the generation of hNPCs and their derivatives in quantities necessary for basic and translational applications.
ContributorsMorgan, Daylin (Author) / Brafman, David (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2018
Description
Calcium imaging is a well-established, non-invasive or minimally technique designed to study the electrical signaling neurons. Calcium regulates the release of gliotransmitters in astrocytes. Analyzing astrocytic calcium transients can provide significant insights into mechanisms such as neuroplasticity and neural signal modulation.
In the past decade, numerous methods have been developed to analyze in-vivo calcium imaging data that involves complex techniques such as overlapping signals segregation and motion artifact correction. The hypothesis used to detect calcium signal is the spatiotemporal sparsity of calcium signal, and these methods are unable to identify the passive cells that are not actively firing during the time frame in the video. Statistics regarding the percentage of cells in each frame of view can be critical for the analysis of calcium imaging data for human induced pluripotent stem cells derived neurons and astrocytes.
The objective of this research is to develop a simple and efficient semi-automated pipeline for analysis of in-vitro calcium imaging data. The region of interest (ROI) based image segmentation is used to extract the data regarding intensity fluctuation caused by calcium concentration changes in each cell. It is achieved by using two approaches: basic image segmentation approach and a machine learning approach. The intensity data is evaluated using a custom-made MATLAB that generates statistical information and graphical representation of the number of spiking cells in each field of view, the number of spikes per cell and spike height.
In the past decade, numerous methods have been developed to analyze in-vivo calcium imaging data that involves complex techniques such as overlapping signals segregation and motion artifact correction. The hypothesis used to detect calcium signal is the spatiotemporal sparsity of calcium signal, and these methods are unable to identify the passive cells that are not actively firing during the time frame in the video. Statistics regarding the percentage of cells in each frame of view can be critical for the analysis of calcium imaging data for human induced pluripotent stem cells derived neurons and astrocytes.
The objective of this research is to develop a simple and efficient semi-automated pipeline for analysis of in-vitro calcium imaging data. The region of interest (ROI) based image segmentation is used to extract the data regarding intensity fluctuation caused by calcium concentration changes in each cell. It is achieved by using two approaches: basic image segmentation approach and a machine learning approach. The intensity data is evaluated using a custom-made MATLAB that generates statistical information and graphical representation of the number of spiking cells in each field of view, the number of spikes per cell and spike height.
ContributorsBhandarkar, Siddhi Umesh (Author) / Brafman, David (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Tian, Xiaojun (Committee member) / Arizona State University (Publisher)
Created2019
Description
Several debilitating neurological disorders, such as Alzheimer's disease, stroke, and spinal cord injury, are characterized by the damage or loss of neuronal cell types in the central nervous system (CNS). Human neural progenitor cells (hNPCs) derived from human pluripotent stem cells (hPSCs) can proliferate extensively and differentiate into the various neuronal subtypes and supporting cells that comprise the CNS. As such, hNPCs have tremendous potential for disease modeling, drug screening, and regenerative medicine applications. However, the use hNPCs for the study and treatment of neurological diseases requires the development of defined, robust, and scalable methods for their expansion and neuronal differentiation. To that end a rational design process was used to develop a vitronectin-derived peptide (VDP)-based substrate to support the growth and neuronal differentiation of hNPCs in conventional two-dimensional (2-D) culture and large-scale microcarrier (MC)-based suspension culture. Compared to hNPCs cultured on ECMP-based substrates, hNPCs grown on VDP-coated surfaces displayed similar morphologies, growth rates, and high expression levels of hNPC multipotency markers. Furthermore, VDP surfaces supported the directed differentiation of hNPCs to neurons at similar levels to cells differentiated on ECMP substrates. Here it has been demonstrated that VDP is a robust growth and differentiation matrix, as demonstrated by its ability to support the expansions and neuronal differentiation of hNPCs derived from three hESC (H9, HUES9, and HSF4) and one hiPSC (RiPSC) cell lines. Finally, it has been shown that VDP allows for the expansion or neuronal differentiation of hNPCs to quantities (>1010) necessary for drug screening or regenerative medicine purposes. In the future, the use of VDP as a defined culture substrate will significantly advance the clinical application of hNPCs and their derivatives as it will enable the large-scale expansion and neuronal differentiation of hNPCs in quantities necessary for disease modeling, drug screening, and regenerative medicine applications.
ContributorsVarun, Divya (Author) / Brafman, David (Thesis advisor) / Nikkhah, Mehdi (Committee member) / Stabenfeldt, Sarah (Committee member) / Arizona State University (Publisher)
Created2016
Description
During the 1960s, the long-standing idea that traits or behaviors could be
explained by natural selection acting on traits that persisted "for the good of the group" prompted a series of debates about group-level selection and the effectiveness with which natural selection could act at or across multiple levels of biological organization. For some this topic remains contentious, while others consider the debate settled, even while disagreeing about when and how resolution occurred, raising the question: "Why have these debates continued?"
Here I explore the biology, history, and philosophy of the possibility of natural selection operating at levels of biological organization other than the organism by focusing on debates about group-level selection that have occurred since the 1960s. In particular, I use experimental, historical, and synthetic methods to review how the debates have changed, and whether different uses of the same words and concepts can lead to different interpretations of the same experimental data.
I begin with the results of a group-selection experiment I conducted using the parasitoid wasp Nasonia, and discuss how the interpretation depends on how one conceives of and defines a "group." Then I review the history of the group selection controversy and argue that this history is best interpreted as multiple, interrelated debates rather than a single continuous debate. Furthermore, I show how the aspects of these debates that have changed the most are related to theoretical content and empirical data, while disputes related to methods remain largely unchanged. Synthesizing this material, I distinguish four different "approaches" to the study of multilevel selection based on the questions and methods used by researchers, and I use the results of the Nasonia experiment to discuss how each approach can lead to different interpretations of the same experimental data. I argue that this realization can help to explain why debates about group and multilevel selection have persisted for nearly sixty years. Finally, the conclusions of this dissertation apply beyond evolutionary biology by providing an illustration of how key concepts can change over time, and how failing to appreciate this fact can lead to ongoing controversy within a scientific field.
explained by natural selection acting on traits that persisted "for the good of the group" prompted a series of debates about group-level selection and the effectiveness with which natural selection could act at or across multiple levels of biological organization. For some this topic remains contentious, while others consider the debate settled, even while disagreeing about when and how resolution occurred, raising the question: "Why have these debates continued?"
Here I explore the biology, history, and philosophy of the possibility of natural selection operating at levels of biological organization other than the organism by focusing on debates about group-level selection that have occurred since the 1960s. In particular, I use experimental, historical, and synthetic methods to review how the debates have changed, and whether different uses of the same words and concepts can lead to different interpretations of the same experimental data.
I begin with the results of a group-selection experiment I conducted using the parasitoid wasp Nasonia, and discuss how the interpretation depends on how one conceives of and defines a "group." Then I review the history of the group selection controversy and argue that this history is best interpreted as multiple, interrelated debates rather than a single continuous debate. Furthermore, I show how the aspects of these debates that have changed the most are related to theoretical content and empirical data, while disputes related to methods remain largely unchanged. Synthesizing this material, I distinguish four different "approaches" to the study of multilevel selection based on the questions and methods used by researchers, and I use the results of the Nasonia experiment to discuss how each approach can lead to different interpretations of the same experimental data. I argue that this realization can help to explain why debates about group and multilevel selection have persisted for nearly sixty years. Finally, the conclusions of this dissertation apply beyond evolutionary biology by providing an illustration of how key concepts can change over time, and how failing to appreciate this fact can lead to ongoing controversy within a scientific field.
ContributorsDimond, Christopher C (Author) / Collins, James P. (Thesis advisor) / Gadau, Juergen (Committee member) / Laubichler, Manfred (Committee member) / Armendt, Brad (Committee member) / Lynch, John (Committee member) / Arizona State University (Publisher)
Created2014