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- Genre: Doctoral Dissertation
- Member of: Theses and Dissertations
Description
Scientists are entrusted with developing novel molecular strategies for effective prophylactic and therapeutic interventions. Antivirals are indispensable tools that can be targeted at viral domains directly or at cellular domains indirectly to obstruct viral infections and reduce pathogenicity. Despite their transformative potential in healthcare, to date, antivirals have been clinically approved to treat only 10 out of the greater than 200 known pathogenic human viruses. Additionally, as obligate intracellular parasites, many virus functions are intimately coupled with host cellular processes. As such, the development of a clinically relevant antiviral is challenged by the limited number of clear targets per virus and necessitates an extensive insight into these molecular processes. Compounding this challenge, many viral pathogens have evolved to evade effective antivirals. Therefore, a means to develop virus- or strain-specific antivirals without detailed insight into each idiosyncratic biochemical mechanism may aid in the development of antivirals against a larger swath of pathogens. Such an approach will tremendously benefit from having the specific molecular recognition of viral species as the lowest barrier. Here, I modify a nanobody (anti-green fluorescent protein) that specifically recognizes non-essential epitopes (glycoprotein M-pHluorin chimera) presented on the extra virion surface of a virus (Pseudorabies virus strain 486). The nanobody switches from having no inhibitory properties (tested up to 50 μM) to ∼3 nM IC50 in in vitro infectivity assays using porcine kidney (PK15) cells. The nanobody modifications use highly reliable bioconjugation to a three-dimensional wireframe deoxyribonucleic acid (DNA) origami scaffold. Mechanistic studies suggest that inhibition is mediated by the DNA origami scaffold bound to the virus particle, which obstructs the internalization of the viruses into cells, and that inhibition is enhanced by avidity resulting from multivalent virus and scaffold interactions. The assembled nanostructures demonstrate negligible cytotoxicity (<10 nM) and sufficient stability, further supporting their therapeutic potential. If translatable to other viral species and epitopes, this approach may open a new strategy that leverages existing infrastructures – monoclonal antibody development, phage display, and in vitro evolution - for rapidly developing novel antivirals in vivo.
ContributorsPradhan, Swechchha (Author) / Hariadi, Rizal (Thesis advisor) / Hogue, Ian (Committee member) / Varsani, Arvind (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2022
Description
Monkeypox virus (MPXV) is an orthopoxvirus that causes smallpox-like disease and has up to a 10% mortality rate, depending on the infectious strain. The global eradication of the smallpox virus has led to the decrease in smallpox vaccinations, which has led to a drastic increase in the number of human MPXV cases. MPXV has been named the most important orthopoxvirus to infect humans since the eradication of smallpox and has been the causative agent of the 2022 world-wide MPXV outbreak. Despite being highly pathogenic, MPXV contains a natural truncation at the N-terminus of its E3 homologue. Vaccinia virus (VACV) E3 protein has two domains: an N- terminus Z-form nucleic acid binding domain (Z-BD) and a C-terminus double stranded RNA binding domain (dsRBD). Both domains are required for pathogenesis, interferon (IFN) resistance, and protein kinase R (PKR) inhibition. The N-terminus is required for evasion of Z-DNA binding protein 1 (ZBP1)-dependent necroptosis. ZBP1 binding to Z- form deoxyribonucleic acid/ribonucleic acid (Z-DNA/RNA) leads to activation of receptor-interacting protein kinase 3 (RIPK3) leading to mixed lineage kinase domain- like (MLKL) phosphorylation, aggregation and cell death. This study investigated how different cell lines combat MPXV infection and how MPXV has evolved ways to circumvent the host response. MPXV is shown to inhibit necroptosis in L929 cells by degrading RIPK3 through the viral inducer of RIPK3 degradation (vIRD) and by inhibiting MLKL aggregation. Additionally, the data shows that IFN treatment efficiently inhibits MPXV replication in a ZBP1-, RIPK3-, and MLKL- dependent manner, but independent of necroptosis. Also, the data suggests that an IFN inducer with a pancaspase or proteasome inhibitor could potentially be a beneficial treatment against MPXV infections. Furthermore, it reveals a link between PKR and pathogen-induced necroptosis that has not been previously described.
ContributorsWilliams, Jacqueline (Author) / Jacobs, Bertram (Thesis advisor) / Langland, Jeffrey (Committee member) / Lake, Douglas (Committee member) / Varsani, Arvind (Committee member) / Arizona State University (Publisher)
Created2022
Description
The topic of this dissertation is the sustainable disposal of waste materials in a way that mitigates risk to human health and the environment. A meta-analysis of organic contaminant concentrations in U.S. sewage sludge highlights the known analytes detected across 106 studies, and juxtaposes these data with a Chinese sludge analysis covering 159 studies, finding that U.S. average concentrations were higher than Chinese concentrations in 26 out of 34 tested organic chemicals. To further investigate the risk that sewage sludge poses when applied on agricultural land mixed with fertilizer as a soil amendment, a sewage sludge risk assessment that for the first time utilized Monte-Carlo simulations was performed to quantify the human health risk of metals present in sewage sludge applied on soils subject to involuntarily ingestion. This study found that while hazard indexes did not indicate a risk to humans for the metals studied, hundreds of other inorganic and organic chemicals are known to be present whose human health risks remain uncertain due to a lack of toxicological data. Among these contaminants are micro- and nanoplastics which contaminate not just sewage sludge but the entire globe. Application of existing models to the world’s oceans showed micro- and nanoplastics to constitute an important component of the total global plastic waste inventory, forecasting peak exposures of aquatic organisms (and by extension human populations) to occur in future years irrespective of what policy options will be implemented. A review of disposal options for sewage sludge illustrates the challenge of dealing with waste streams containing persistent or even indestructible contaminants such as perfluorinated organics, mass-produced fossil-fuel derived consumer plastics, and extensively mined toxic metals. The work presented here details the risks, both avoidable and unavoidable, that are present in the disposal of sewage sludge and plastics. The information presented in this dissertation may inform regulatory actions to promote environmentally responsible disposal and reuse of sewage sludge and highlights the need for industry to transition to the production of more sustainable plastics in order to reduce and ultimately eliminate sources of persistent long-term environmental pollution and their associated adverse human health and ecosystem impacts.
ContributorsSteele, Joshua (Author) / Halden, Rolf U (Thesis advisor) / Mascaro, Giuseppe (Committee member) / Meier, Madeline (Committee member) / Arizona State University (Publisher)
Created2022
Description
Traditional public health strategies for assessing human behavior, exposure, and activity are considered resource-exhaustive, time-consuming, and expensive, warranting a need for alternative methods to enhance data acquisition and subsequent interventions. This dissertation critically evaluated the use of wastewater-based epidemiology (WBE) as an inclusive and non-invasive tool for conducting near real-time population health assessments. A rigorous literature review was performed to gauge the current landscape of WBE to monitor for biomarkers indicative of diet, as well as exposure to estrogen-mimicking endocrine disrupting (EED) chemicals via route of ingestion. Wastewater-derived measurements of phytoestrogens from August 2017 through July 2019 (n = 156 samples) in a small sewer catchment revealed seasonal patterns, with highest average per capita consumption rates in January through March of each year (2018: 7.0 ± 2.0 mg d-1; 2019: 8.2 ± 2.3 mg d-1) and statistically significant differences (p = 0.01) between fall and winter (3.4 ± 1.2 vs. 6.1 ± 2.9 mg d-1; p ≤ 0.01) and spring and summer (5.6 ± 2.1 vs. 3.4 ± 1.5 mg d-1; p ≤ 0.01). Additional investigations, including a human gut microbial composition analysis of community wastewater, were performed to support a methodological framework for future implementation of WBE to assess population-level dietary behavior. In response to the COVID-19 global pandemic, a high-frequency, high-resolution sample collection approach with public data sharing was implemented throughout the City of Tempe, Arizona, and analyzed for SARS-CoV-2 (E gene) from April 2020 through March 2021 (n = 1,556 samples). Results indicate early warning capability during the first wave (June 2020) compared to newly reported clinical cases (8.5 ± 2.1 days), later transitioning to a slight lagging indicator in December/January 2020-21 (-2.0 ± 1.4 days). A viral hotspot from within a larger catchment area was detected, prompting targeted interventions to successfully mitigate community spread; reinforcing the importance of sample collection within the sewer infrastructure. I conclude that by working in tandem with traditional approaches, WBE can enlighten a comprehensive understanding of population health, with methods and strategies implemented in this work recommended for future expansion to produce timely, actionable data in support of public health.
ContributorsBowes, Devin Ashley (Author) / Halden, Rolf U (Thesis advisor) / Krajmalnik-Brown, Rosa (Thesis advisor) / Conroy-Ben, Otakuye (Committee member) / Varsani, Arvind (Committee member) / Whisner, Corrie (Committee member) / Arizona State University (Publisher)
Created2022
Description
This dissertation applies wastewater-based epidemiology (WBE) to aqueous process flows to gauge the public health status concerning exposure and potential abuse of pharmaceuticals, antimicrobials, and narcotics. The masses of emerging contaminants emitted into Indian aquatic and terrestrial environments were the highest for open defecation (17 ± 12 mt/d), with non-steroidal anti-inflammatory drugs dominating environmental loading (14 ± 10 mt/d), followed by antibiotics, antimicrobials, phthalates and miscellaneous pharmaceuticals (Chapter 2). Fourteen wastewater treatment plants sampled across the U.S. had a combined average mass loading of 71 ± 12 µg/d/capita for the antimicrobials triclosan and triclocarban, with paraben compounds contributing 19 ± 5 µg/d/US capita. Risk models showed unfavorable hazard quotients (HQ>1) for sensitive aquatic organisms (algae, zebra fish and rainbow trout) from predicted exposures to antimicrobials of alternative use, i.e., chlorhexidine and benzalkonium chloride (Chapter 3). Substances subject to licit and illicit use, monitored by WBE in a medium-sized southwestern U.S. city before and during COVID-19-related lockdowns, showed the highest mass loads for cocaine and its major metabolite benzoylecgonine (2,207 total), methadone and its major metabolite 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (197), parent mitragynine (60), oxycodone and its major metabolite noroxycodone (48), heroin and its major metabolite 6-acetylmorphine (45), and parent codeine (37) in mg/1,000 capita/day. Heroin use during the lockdown increased ~10-fold relative to the pre-lockdown baseline, whereas oxycodone and codeine mass loading decreased 5-fold and 2.5-fold, respectively (Chapter 4). Experiments elucidating the stability of stress hormones and their metabolites as a function of temperature and in-sewer residence time revealed a rapid degradation to completion over 24 hours at 35°C, whereas lower temperatures of 25°C and 15°C were found to allow for successful tracking of indicators of stress at the population level; statistically significant differences in stress hormone decay rates were observed due to geographic locations at 25°C (p=0.009) but not due to redox conditions in the sewer pipe (Chapter 5). This thesis demonstrated the successful application of WBE for studying population health frequently and inexpensively, with the limitation that a lack of centralized wastewater infrastructure in developing countries may create barriers for at-risk populations to access and utilize this novel technology (Chapter 6).
ContributorsKelkar, Varun Pushkaraj (Author) / Halden, Rolf U (Thesis advisor) / Hamilton, Kerry A (Committee member) / Conroy-Ben, Otakuye (Committee member) / Arizona State University (Publisher)
Created2021
Description
Poxviruses such as monkeypox virus (MPXV) are emerging zoonotic diseases. Compared to MPXV, Vaccinia virus (VACV) has reduced pathogenicity in humans and can be used as a partially protective vaccine against MPXV. While most orthopoxviruses have E3 protein homologues with highly similar N-termini, the MPXV homologue, F3, has a start codon mutation leading to an N-terminal truncation of 37 amino acids. The VACV protein E3 consists of a dsRNA binding domain in its C-terminus which must be intact for pathogenicity in murine models and replication in cultured cells. The N-terminus of E3 contains a Z-form nucleic acid (ZNA) binding domain and is also required for pathogenicity in murine models. Poxviruses produce RNA transcripts that extend beyond the transcribed gene which can form double-stranded RNA (dsRNA). The innate immune system easily recognizes dsRNA through proteins such as protein kinase R (PKR). After comparing a vaccinia virus with a wild-type E3 protein (VACV WT) to one with an E3 N-terminal truncation of 37 amino acids (VACV E3Δ37N), phenotypic differences appeared in several cell lines. In HeLa cells and certain murine embryonic fibroblasts (MEFs), dsRNA recognition pathways such as PKR become activated during VACV E3Δ37N infections, unlike VACV WT. However, MPXV does not activate PKR in HeLa or MEF cells. Additional investigation determined that MPXV produces less dsRNA than VACV. VACV E3Δ37N was made more similar to MPXV by selecting mutants that produce less dsRNA. By producing less dsRNA, VACV E3Δ37N no longer activated PKR in HeLa or MEF cells, thus restoring the wild-type phenotype. Furthermore, in other cell lines such as L929 (also a murine fibroblast) VACV E3Δ37N, but not VACV WT infection leads to activation of DNA-dependent activator of IFN-regulatory factors (DAI) and induction of necroptotic cell death. The same low dsRNA mutants demonstrate that DAI activation and necroptotic induction is independent of classical dsRNA. Finally, investigations of spread in an animal model and replication in cell lines where both the PKR and DAI pathways are intact determined that inhibition of both pathways is required for VACV E3Δ37N to replicate.
ContributorsCotsmire, Samantha (Author) / Jacobs, Bertram L (Thesis advisor) / Varsani, Arvind (Committee member) / Hogue, Brenda (Committee member) / Haydel, Shelley (Committee member) / Arizona State University (Publisher)
Created2021
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
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
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
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