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Description
Precise modulation of gene expression is essential for proper tissue and cell-specific differentiation and function. Multiple distinct post-transcriptional regulatory mechanisms, such as miRNA (microRNA)-based regulation and alternative polyadenylation (APA), are an intrinsic part of this modulation and orchestrate intricate pathways to achieve and maintain balanced gene expression.MiRNA-based regulation and APA function through sequence motifs located in the 3’ Untranslated Region (3’UTR) of mRNA transcripts. MiRNAs are short (~22 nt) non-coding RNA molecules that bind target sequences within the 3’UTR of an mRNA transcript, inhibiting its translation or promoting its degradation. APA occurs during RNA transcription termination and leads to the preparation of mature mRNAs with different 3’UTR lengths, allowing shorter 3’UTRs to bypass miRNA regulation.
In addition to these two post-transcriptional forms of regulation, co-transcriptional mechanisms such as alternative RNA splicing, which produces distinct gene products from a precursor mRNA, are also important in controlling gene expression.
While miRNA-based regulation, APA, and alternative RNA splicing are important regulatory mechanisms, there is a lack of comprehensive understanding of how they interact and communicate with each other. This thesis studies these three forms of gene regulation in the nematode C. elegans, with the goal of extracting rules and mechanisms used by each of them in development to establish and maintain somatic tissue identity.
After isolating miRNA targets in multiple C. elegans somatic tissues, it was found that miRNAs can modulate the abundance of hnRNPs and SR proteins, which are known to control alternative RNA splicing in a dosage-dependent manner.To identify tissue-specific miRNAs, a nuclear fluorescent cell sorting (FACS)-based methodology named Nuc-Seq, was developed to isolate and sequence tissue-specific miRNAs from body muscle tissue. Nuc-Seq identified 2,848 muscle-specific protein-coding genes and 16 body muscle-specific miRNAs. This data was used to develop a high-quality body muscle-specific miRNA-APA Interactome which allows studies in regulatory processes in detail.
Taken together, this work highlights some of the complexity of pre- and post-transcriptional gene regulation and sheds light on how miRNA-based regulation, APA, and alternative RNA splicing are interconnected and are responsible for the establishment and maintenance of tissue identity.
ContributorsSchorr, Anna L (Author) / Mangone, Marco (Thesis advisor) / Harris, Robin (Committee member) / Sharma, Shalini (Committee member) / Varsani, Arvind (Committee member) / Arizona State University (Publisher)
Created2023
Description
Despite the prevalence of coyotes (Canis latrans) little is known about the viruses associated with this species. To assess the extent of viral research that has been conducted on coyotes, a literature review was performed. Over the last six decades, there have been many viruses that have been identified infecting coyotes. The pathology of some cases implies that infection is rare and lethal while others have been demonstrated to be endemic to coyotes. In addition, the majority of the prior analyses were done through serological assays that were limited to investigating target viruses. To help expand what is known about coyote-virus dynamics, viral assays were conducted on coyote scat. The samples were collected as part of transects established along the Salt River near Phoenix, Arizona, United States (USA). The recovered viral genomes were clustered with other deoxynucleic acid (DNA) viruses and analyzed to determine phylogeny and genetic identity. From the recovered viral genomes, there are two novel circoviruses, one novel naryavirus, five unclassified cressdnaviruses, and two previously identified species of anelloviruses from the Wawtorquevirus genus. For these viruses, new phylogenies for their groups and pairwise identity plots have been generated. These figures give insight into the potential hosts and the evolutionary history. In the case of the anelloviruses, they likely derived from a wood rat (Neotoma) host, given the anellovirus family’s host specificity and its similarity to another viral genome derived from a wood rat in Arizona, USA. Of the recovered circovirus genomes, one is associated with a viral isolate collected from a dust sample in Arizona, USA. The second circovirus species identified is within a clade that consists of rodent associated circoviruses and canine circovirus. Other recovered genomes expand clusters of unclassified cressdnaviruses. The recovered genomes support further genomic analysis. These findings help support the notion that there is a wealth of viral information to be identified from animals like coyotes. By understanding the viruses that coyotes are associated with, it is possible to better understand the viral impact on the urban environment, domesticated animals, and wildlife in general.
ContributorsHess, Savage Cree (Author) / Varsani, Arvind (Thesis advisor) / Kraberger, Simona (Committee member) / Upham, Nathan S (Committee member) / Arizona State University (Publisher)
Created2023
Description
Alpha herpesviruses are a family of neuroinvasive viruses that infect multiplevertebrate species. Alpha herpesviruses are responsible for human and livestock infections, most notably Herpes Simplex Virus (HSV), Varicella Zoster virus (VZV), and Pseudorabies Virus (PRV). PRV is a potent swine virus that can infect other mammals, and results in lethal encephalitis that can be devastating to livestock and of great financial expense to farmers. HSV, types 1 and 2, and VZV are widespread throughout the global human population, with estimates of the HSV-1 burden at about 60% of people worldwide. The hallmark of alpha herpesvirus infection is a persistent, lifelong infection that can reactivate throughout the lifespan of the host. Currently, the precise mechanisms of how these viruses undergo intracellular trafficking to emerge from the infected cell in epithelial tissues is not well understood. Many insights have been made with PRV in animal neurons, both in culture systems and animal models, about the viral genes and host factors involved in these processes. However, understanding of these mechanisms, and the interplay between viral and host proteins, in the human pathogen HSV-1 is even more lacking. Using recombinant fluorescent virus strains of HSV-1 and Total Internal Reflection Microscopy to image the transport of mature viral progeny in epithelial cells, it was determined that the egress of HSV-1 uses constitutive cellular secretory pathways. Specifically, the viral progeny traffic from the trans-Golgi network to the site of exocytosis at the plasma membrane via Rab6a secretory vesicles. This work will contribute to the understanding of how alpha herpesviruses complete their lifecycles in host cells, particularly at the sites where infection initially occurs and can spread to a new organism. Knowledge of these processes may lead to the development of therapeutics or prophylactics to reduce the burden of these viruses.
ContributorsBergeman, Melissa Hope (Author) / Hogue, Ian B (Thesis advisor) / Hogue, Brenda (Committee member) / Roberson, Robert (Committee member) / Varsani, Arvind (Committee member) / Arizona State University (Publisher)
Created2023
Description
Maternal morbidity and mortality rates in the United States continues to rise, with a wide range of contributing factors such as mental illness, cardiovascular disease and systemic inequality. This metastudy provides a holistic view of the research that has been published on the issue of U.S. maternal healthcare from 2000-2022. The patterns of publications on specific topics over time can tell us what is perceived as a current major cause by physicians, public leaders, researchers, and the public. A deeper dive into systemic inequality as a cause of maternal morbidity and mortality highlights it as a major contributor to these high rates, but that progress is slowly being made through the implementation of detection and prevention tactics, as well as accessible prenatal programs and care.
ContributorsRettig, Lelia (Author) / Amdam, Gro (Thesis director) / Bang, Christofer (Committee member) / Barrett, The Honors College (Contributor) / School of Human Evolution & Social Change (Contributor) / School of Life Sciences (Contributor)
Created2023-05
DescriptionA
ContributorsLund, Michael (Author) / Varsani, Arvind (Thesis advisor) / Upham, Nathan (Committee member) / Harris, Robin (Committee member) / Arizona State University (Publisher)
Created2023
Description
The human gut microbiome is associated with health outcomes including gastrointestinal and metabolic health, autoimmune disease and cancer. However, the role of the microbiome in many disease processes, including in the preterm gastrointestinal tract and female genital tract, has yet to be defined. Further, the diverse community of viruses within the microbiome (the virome) is understudied compared to bacteria. Here, I examine the microbiome and virome in specific disease models that are poorly understood: necrotizing enterocolitis (NEC), discordant HIV shedding in women living with HIV (WHLIV), female genital tract inflammation and gammaherpesvirus infection. Specifically, I examined the gut virome longitudinally in a cohort of preterm infants at risk for NEC; the female genital tract (FGT) microbiome and virome longitudinally in a cohort of WLHIV from Lima, Peru; the FGT virome in women from Phoenix, Arizona with differing levels of genital inflammation and different microbiome compositions; and the gut microbiome in murine gammaherpesvirus 68 (MHV68) infection. Further, I contributed to research responding to the spread of SARS-CoV-2 in Arizona. I found that 1) gut virome beta diversity decreased before NEC onset in preterm infants, suggesting a role for the virome in NEC; 2) FGT microbiome instability was associated with discordant HIV shedding, while FGT virome composition changed in association with ART duration and immune recovery; 3) FGT virome composition was associated with inflammation and microbiome composition; and 4) MHV68 infection outcomes were independent of microbiome perturbation, which may reflect environmental influences. The results of this research advance understanding of the microbiome and virome in these specific disease processes, and support further investigation of the microbiome and virome in preterm infant gastrointestinal health and FGT health, as well as environmental effects in microbiome research.
ContributorsKaelin, Emily (Author) / Lim, Efrem (Thesis advisor) / Varsani, Arvind (Committee member) / Jacobs, Bertram (Committee member) / McFadden, Grant (Committee member) / Rahman, Masmudur (Committee member) / Arizona State University (Publisher)
Created2024
Description
Arachnids belong to the phylum Arthropoda, the largest phylum in the animal kingdom. Ticks are blood-feeding arachnids that vector numerous pathogens of significant medical and veterinary importance, while scorpions have become a common concern in urban desert cities due to the high level of toxicity in their venom. To date, viruses associated with arachnids have been under sampled and understudied. Here viral metagenomics was used to explore the diversity of viruses present in ticks and scorpions. American dog ticks (Dermacentor variabilis) and blacklegged ticks (Ixodes scapularis) were collected in Pennsylvania while one hairy scorpion (Hadrurus arizonensis) and four bark scorpions (Centruroides sculpturatus) were collected in Phoenix. Novel viral genomes described here belong to the families Polyomaviridae, Anelloviridae, Genomoviridae, and a newly proposed family, Arthropolviridae.
Polyomaviruses are non-enveloped viruses with a small, circular double-stranded DNA (dsDNA) genomes that have been identified in a variety of mammals, birds and fish and are known to cause various diseases. Arthropolviridae is a proposed family of circular, large tumor antigen encoding dsDNA viruses that have a unidirectional genome organization. Genomoviruses and anelloviruses are ssDNA viruses that have circular genomes ranging in size from 2–2.4 kb and 2.1–3.8 kb, respectively. Genomoviruses are ubiquitous in the environment, having been identified in a wide range of animal, plant and environmental samples, while anelloviruses have been associated with a plethora of animals.
Here, 16 novel viruses are reported that span four viral families. Eight novel polyomaviruses were recovered from bark scorpions, three arthropolviruses were recovered from dog ticks and one arthropolvirus from a hairy scorpion. Viruses belonging to the families Polyomaviridae and Arthropolviridae are highly divergent. This is the first more extensive study of these viruses in arachnids. Three genomoviruses were recovered from both dog and deer ticks and one anellovirus was recovered from deer ticks, which are the first records of these viruses being recovered from ticks. This work highlights the diversity of dsDNA and ssDNA viruses in the arachnid population and emphasizes the importance of performing viral surveys on these populations.
Polyomaviruses are non-enveloped viruses with a small, circular double-stranded DNA (dsDNA) genomes that have been identified in a variety of mammals, birds and fish and are known to cause various diseases. Arthropolviridae is a proposed family of circular, large tumor antigen encoding dsDNA viruses that have a unidirectional genome organization. Genomoviruses and anelloviruses are ssDNA viruses that have circular genomes ranging in size from 2–2.4 kb and 2.1–3.8 kb, respectively. Genomoviruses are ubiquitous in the environment, having been identified in a wide range of animal, plant and environmental samples, while anelloviruses have been associated with a plethora of animals.
Here, 16 novel viruses are reported that span four viral families. Eight novel polyomaviruses were recovered from bark scorpions, three arthropolviruses were recovered from dog ticks and one arthropolvirus from a hairy scorpion. Viruses belonging to the families Polyomaviridae and Arthropolviridae are highly divergent. This is the first more extensive study of these viruses in arachnids. Three genomoviruses were recovered from both dog and deer ticks and one anellovirus was recovered from deer ticks, which are the first records of these viruses being recovered from ticks. This work highlights the diversity of dsDNA and ssDNA viruses in the arachnid population and emphasizes the importance of performing viral surveys on these populations.
ContributorsSchmidlin, Kara (Author) / Varsani, Arvind (Thesis advisor) / Van Doorslaer, Koenraad (Committee member) / Stenglein, Mark (Committee member) / Arizona State University (Publisher)
Created2019
Description
Neurotoxicology has historically focused on substances that directly damage nervous tissue. Behavioral assays that test sensory, cognitive, or motor function are used to identify neurotoxins. But, the outcomes of behavioral assays may also be influenced by the physiological status of non-neural organs. Therefore, toxin induced damage to non- neural organs may contribute to behavioral modifications. Heavy metals and metalloids are persistent environmental pollutants and induce neurological deficits in multiple organisms. However, in the honey bee, an important insect pollinator, little is known about the sublethal effects of heavy metal and metalloid toxicity though they are exposed to these toxins chronically in some environments. In this thesis I investigate the sublethal effects of copper, cadmium, lead, and selenium on honey bee behavior and identify potential mechanisms mediating the behavioral modifications. I explore the honey bees’ ability to detect these toxins, their sensory perception of sucrose following toxin exposure, and the effects of toxin ingestion on performance during learning and memory tasks. The effects depend on the specific metal. Honey bees detect and reject copper containing solutions, but readily consume those contaminated with cadmium and lead. And, exposure to lead may alter the sensory perception of sucrose. I also demonstrate that acute selenium exposure impairs learning and long-term memory formation or recall. Localizing selenium accumulation following chronic exposure reveals that damage to non-neural organs and peripheral sensory structures is more likely than direct neurotoxicity. Probable mechanisms include gut microbiome alterations, gut lining
damage, immune system activation, impaired protein function, or aberrant DNA methylation. In the case of DNA methylation, I demonstrate that inhibiting DNA methylation dynamics can impair long-term memory formation, while the nurse-to- forager transition is not altered. These experiments could serve as the bases for and reference groups of studies testing the effects of metal or metalloid toxicity on DNA methylation. Each potential mechanism provides an avenue for investigating how neural function is influenced by the physiological status of non-neural organs. And from an ecological perspective, my results highlight the need for environmental policy to consider sublethal effects in determining safe environmental toxin loads for honey bees and other insect pollinators.
damage, immune system activation, impaired protein function, or aberrant DNA methylation. In the case of DNA methylation, I demonstrate that inhibiting DNA methylation dynamics can impair long-term memory formation, while the nurse-to- forager transition is not altered. These experiments could serve as the bases for and reference groups of studies testing the effects of metal or metalloid toxicity on DNA methylation. Each potential mechanism provides an avenue for investigating how neural function is influenced by the physiological status of non-neural organs. And from an ecological perspective, my results highlight the need for environmental policy to consider sublethal effects in determining safe environmental toxin loads for honey bees and other insect pollinators.
ContributorsBurden, Christina Marie (Author) / Amdam, Gro (Thesis advisor) / Smith, Brian H. (Thesis advisor) / Gallitano-Mendel, Amelia (Committee member) / Harrison, Jon (Committee member) / Vu, Eric (Committee member) / Arizona State University (Publisher)
Created2016
Description
One of the single-most insightful, and visionary talks of the 20th century, “There’s plenty of room at the bottom,” by Dr. Richard Feynman, represented a first foray into the micro- and nano-worlds of biology and chemistry with the intention of direct manipulation of their individual components. Even so, for decades there has existed a gulf between the bottom-up molecular worlds of biology and chemistry, and the top-down world of nanofabrication. Creating single molecule nanoarrays at the limit of diffraction could incentivize a paradigm shift for experimental assays. However, such arrays have been nearly impossible to fabricate since current nanofabrication tools lack the resolution required for precise single-molecule spatial manipulation. What if there existed a molecule which could act as a bridge between these top-down and bottom-up worlds?
At ~100-nm, a DNA origami macromolecule represents one such bridge, acting as a breadboard for the decoration of single molecules with 3-5 nm resolution. It relies on the programmed self-assembly of a long, scaffold strand into arbitrary 2D or 3D structures guided via approximately two hundred, short, staple strands. Once synthesized, this nanostructure falls in the spatial manipulation regime of a nanofabrication tool such as electron-beam lithography (EBL), facilitating its high efficiency immobilization in predetermined binding sites on an experimentally relevant substrate. This placement technology, however, is expensive and requires specialized training, thereby limiting accessibility.
The work described here introduces a method for bench-top, cleanroom/lithography-free, DNA origami placement in meso-to-macro-scale grids using tunable colloidal nanosphere masks, and organosilane-based surface chemistry modification. Bench-top DNA origami placement is the first demonstration of its kind which facilitates precision placement of single molecules with high efficiency in diffraction-limited sites at a cost of $1/chip. The comprehensive characterization of this technique, and its application as a robust platform for high-throughput biophysics and digital counting of biomarkers through enzyme-free amplification are elucidated here. Furthermore, this technique can serve as a template for the bottom-up fabrication of invaluable biophysical tools such as zero mode waveguides, making them significantly cheaper and more accessible to the scientific community. This platform has the potential to democratize high-throughput single molecule experiments in laboratories worldwide.
At ~100-nm, a DNA origami macromolecule represents one such bridge, acting as a breadboard for the decoration of single molecules with 3-5 nm resolution. It relies on the programmed self-assembly of a long, scaffold strand into arbitrary 2D or 3D structures guided via approximately two hundred, short, staple strands. Once synthesized, this nanostructure falls in the spatial manipulation regime of a nanofabrication tool such as electron-beam lithography (EBL), facilitating its high efficiency immobilization in predetermined binding sites on an experimentally relevant substrate. This placement technology, however, is expensive and requires specialized training, thereby limiting accessibility.
The work described here introduces a method for bench-top, cleanroom/lithography-free, DNA origami placement in meso-to-macro-scale grids using tunable colloidal nanosphere masks, and organosilane-based surface chemistry modification. Bench-top DNA origami placement is the first demonstration of its kind which facilitates precision placement of single molecules with high efficiency in diffraction-limited sites at a cost of $1/chip. The comprehensive characterization of this technique, and its application as a robust platform for high-throughput biophysics and digital counting of biomarkers through enzyme-free amplification are elucidated here. Furthermore, this technique can serve as a template for the bottom-up fabrication of invaluable biophysical tools such as zero mode waveguides, making them significantly cheaper and more accessible to the scientific community. This platform has the potential to democratize high-throughput single molecule experiments in laboratories worldwide.
ContributorsShetty, Rishabh Manoj (Author) / Hariadi, Rizal F (Thesis advisor) / Gopinath, Ashwin (Committee member) / Varsani, Arvind (Committee member) / Nikkhah, Mehdi (Committee member) / Tillery, Stephen H (Committee member) / Hu, Ye (Committee member) / Arizona State University (Publisher)
Created2019
Description
The human papillomavirus (HPV) is a double-stranded DNA virus responsible for causing upwards of 80% of head and neck cancers in the oropharyngeal region. Current treatments, including surgery, chemotherapy, and/or radiation, are aggressive and elicit toxic effects. HPV is a pathogen that expresses viral-specific oncogenic proteins that play a role in cancer progression. These proteins may serve as potential targets for immunotherapeutic applications. Engineered T cell receptor (TCR) therapy may be an advantageous approach for HPV-associated cancers. In TCR therapy, TCRs are modified to express a receptor that is specific to an immunogenic antigen (part of the virus/cancer capable of eliciting an immune response). Since HPV-associated oropharyngeal cancers typically express unique viral proteins, it is important to identify the TCRs capable of recognizing these proteins. Evidence supports that head and neck cancers typically experience high levels of immune cell infiltration and are subsequently associated with increased survival rates. Most of the immune cell infiltrations in HPV+ HNSCC are CD8+ T lymphocytes, drawing attention to their prospective use in cellular immunotherapies. While TCRs are highly specific, the TCR repertoire is extremely diverse; enabling the immune system to fight off numerous pathogens. In project 1, I review approaches to analyzing TCR diversity and explore the use of DNA origami in retrieving paired TCR sequences from a population. The results determine that DNA origami can be used within a monoclonal population but requires further optimization before being applied in a polyclonal setting. In project 2, I investigate HPV-specific T-cell dysfunction; I detect low frequency HPV-specific CD8+ T cells, determine that they are tumor specific, and show that HPV+HNSCC patients exhibit increased epitope-specific levels of CD8+T cell exhaustion. In project 3, I apply methods to expand and isolate TCRαβ sequences derived from donors stimulated with a previously identified HPV epitope. Single-cell analysis provide ten unique TCRαβ pairs with corresponding CDR3 sequences that may serve as therapeutic candidates. This thesis contributes to fundamental immunology by contributing to the knowledge of T cell dysfunction within HPV+HNSCC and further reveals TCR gene usage within an HPV stimulated population, thus identifying potential TCR pairs for adoptive cell therapies.
ContributorsUlrich, Peaches Rebecca (Author) / Anderson, Karen S (Thesis advisor) / Lake, Douglas (Committee member) / Maley, Carlo (Committee member) / Varsani, Arvind (Committee member) / Arizona State University (Publisher)
Created2020