Start to Signal: A Novel Approach to Isothermal Assay Design for Point-of-care Detection of Emerging Pathogens

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Description
Emerging pathogens present several challenges to medical diagnostics. Primarily, the exponential spread of a novel pathogen through naïve populations require a rapid and overwhelming diagnostic response at the site of outbreak. While point-of-care (PoC) platforms have been developed for detection…

Emerging pathogens present several challenges to medical diagnostics. Primarily, the exponential spread of a novel pathogen through naïve populations require a rapid and overwhelming diagnostic response at the site of outbreak. While point-of-care (PoC) platforms have been developed for detection of antigens, serologic responses, and pathogenic genomes, only nucleic acid diagnostics currently have the potential to be developed and manufactured within weeks of an outbreak owing to the speed of next-generation sequencing and custom DNA synthesis. Among nucleic acid diagnostics, isothermal amplification strategies are uniquely suited for PoC implementation due to their simple instrumentation and lack of thermocycling requirement. Unfortunately, isothermal strategies are currently prone to spurious nonspecific amplification, hindering their specificity and necessitating extensive empirical design pipelines that are both time and resource intensive. In this work, isothermal amplification strategies are extensively compared for their feasibility of implementation in outbreak response scenarios. One such technology, Loop-mediated Amplification (LAMP), is identified as having high-potential for rapid development and PoC deployment. Various approaches to abrogating nonspecific amplification are described including a novel in silico design tool based on coarse-grained simulation of interactions between thermophilic DNA polymerase and DNA strands in isothermal reaction conditions. Nonspecific amplification is shown to be due to stabilization of primer secondary structures by high concentrations of Bst DNA polymerase and a mechanism of micro-complement-mediated cross-priming is demonstrated as causal via nanopore sequencing of nonspecific reaction products. The resulting computational model predicts primer set background in 64% of 67 test assays and its usefulness is illustrated further by determining problematic primers in a West Nile Virus-specific LAMP primer set and optimizing primer 3’ nucleotides to eliminate micro-complements within the reaction, resulting in inhibition of background accumulation. Finally, the emergence of Orthopox monkeypox (MPXV) as a recurring threat is discussed and SimCycle is utilized to develop a novel technique for clade-specific discrimination of MPXV based on bridging viral genomic rearrangements (Bridging LAMP). Bridging LAMP is implemented in a 4-plex microfluidic format and demonstrates 100% sensitivity in detection of 100 copies of viral lysates and 45 crude MPXV-positive patient samples collected during the 2022 Clade IIb outbreak.
Date Created
2023
Agent

The Function of Tyramine within the Male Reproductive System

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Description
Male reproductive dysfunction accounts for almost half of male infertility cases, yet the signaling mechanisms involved in the male reproductive system remain unclear. Although the exact cause of male reproductive dysfunction varies, obtaining a better understanding of the modulators of…

Male reproductive dysfunction accounts for almost half of male infertility cases, yet the signaling mechanisms involved in the male reproductive system remain unclear. Although the exact cause of male reproductive dysfunction varies, obtaining a better understanding of the modulators of smooth muscle contractions may provide new targets for the treatment of male reproductive conditions. The male reproductive tract, consisting of the testes, epididymis, vas deferens, and penis, is lined with innervated smooth muscle fibers that transport spermatozoa through the system. Contractions of these smooth muscle fibers can be modulated by neurotransmitters and hormones, like dopamine and norepinephrine, as well as biogenic amines. The focus of this study is on the biogenic amine tyramine, which is produced by the breakdown of tyrosine via decarboxylation. Tyramine has been shown to modulate vasoconstriction and increase blood pressure due to its effect on smooth muscle contractions. This study has found that tyramine localizes in male reproductive tissues and modulates smooth muscle contractions. Age and environment were also found to play a significant role in the expression of tyramine and its associated receptor, TAAR1.
Date Created
2023
Agent

The Mechanisms of Alpha Herpesvirus Intracellular Trafficking and Exocytosis

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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…

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.
Date Created
2023
Agent

Investigating Tyramine’s Role in the Mouse Uterine Horn

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Description
Human preterm labor is the single most significant issue in modern obstetrics andgynecology, affecting ten percent of pregnancies, constituting the leading cause of infant death, and contributing significantly to chronic childhood disease. Obstetricians and reproductive scientists are faced with the…

Human preterm labor is the single most significant issue in modern obstetrics andgynecology, affecting ten percent of pregnancies, constituting the leading cause of infant death, and contributing significantly to chronic childhood disease. Obstetricians and reproductive scientists are faced with the major challenge of trying to increase the understanding of the complex molecular and cellular signals that regulate uterine activity during human pregnancy and labor. Even though preterm labor accounts for a large portion of perinatal mortality and morbidity, there still is not an effective therapeutic strategy for the treatment or prevention of preterm labor. This dissertation presents tyramine as an alternative modulator of uterine activity. In this dissertation the aims were as follows: 1) to investigate the localization of tyramine and trace amine associated receptor 1 (TAAR1) in the mouse uterine horn using immunohistochemistry as well as confirm the presence of tyramine in the uterine tissue using high performance liquid chromatography, 2) identify which TAAR 1-9 subtypes were present in the mouse uterine horn using RT-qPCR, 3) investigate ultrastructural differences in the mouse uterine horn following tyramine and dopamine treatment using transmission electron microscopy and 4) investigate pinopod ultrastructure as well as pinopod ultrastructural differences following tyramine and dopamine treatment. The research presented in this dissertation showed: 1) tyramine has very specific localization in the mouse endometrium, mainly in the uterine glands, TAAR1 is localized all throughout the perimetrium, myometrium and endometrium, and that tyramine was confirmed and quantified using HPLC, 2) TAAR 1- 9 genes are expressed in trace levels in the mouse uterine horn, 3) tyramine influences changes in endometrial ultrastructure, and 4) tyramine influences changes in pinopod ultrastructure. Ultimately these findings can help with identifying novel treatment options not only for spontaneous preterm labor contractions but also for other uterine related disorders.
Date Created
2023
Agent

Appearance-Based Shame In Medicine: How It Alters Patient Perception and Future Care

Description

Health service quality is understood to be a crucial determinant in successful patient-physician encounters and patient health. One common feeling that patients have reported experiencing during appointments is shame. We hypothesized that patients who experience appearance-based shame during an appointment…

Health service quality is understood to be a crucial determinant in successful patient-physician encounters and patient health. One common feeling that patients have reported experiencing during appointments is shame. We hypothesized that patients who experience appearance-based shame during an appointment are not likely to return to the same physician and that patients who do not experience appearance-based shame are likely to return to the same physician. This was assessed by conducting an anonymous online survey of 13 questions that served to establish a general foundation for understanding the participants' physical characteristics such as race, age, weight, and gender identity as well as their overall patient-physician relationship and experiences of shame, if applicable. 119 participants were recruited from Arizona State University and a case study was performed individually for five participants of interest. The data analyzed from this study suggests that while appearance-based shame does exist in healthcare spaces, it is not a significant determining factor in patients returning to their physicians. In addition, there was no significant evidence to suggest that patients who do not experience appearance-based shame are either likely or more likely to return to their physician. We hypothesize this could be due to confounding variables such as convenience, accessibility, or insurance limitations which patients may prioritize over feeling ashamed during an appointment. However, more research needs to be conducted to confirm these hypotheses.

Date Created
2023-05
Agent

Appearance-Based Shame In Medicine: How It Alters Patient Perception and Future Care

Description

Health service quality is understood to be a crucial determinant in successful patient-physician encounters and patient health. One common feeling that patients have reported experiencing during appointments is shame. We hypothesized that patients who experience appearance-based shame during an appointment…

Health service quality is understood to be a crucial determinant in successful patient-physician encounters and patient health. One common feeling that patients have reported experiencing during appointments is shame. We hypothesized that patients who experience appearance-based shame during an appointment are not likely to return to the same physician and that patients who do not experience appearance-based shame are likely to return to the same physician. This was assessed by conducting an anonymous online survey of 13 questions that served to establish a general foundation for understanding the participants' physical characteristics such as race, age, weight, and gender identity as well as their overall patient-physician relationship and experiences of shame, if applicable. 119 participants were recruited from Arizona State University and a case study was performed individually for five participants of interest. The data analyzed from this study suggests that while appearance-based shame does exist in healthcare spaces, it is not a significant determining factor in patients returning to their physicians. In addition, there was no significant evidence to suggest that patients who do not experience appearance-based shame are either likely or more likely to return to their physician. We hypothesize this could be due to confounding variables such as convenience, accessibility, or insurance limitations which patients may prioritize over feeling ashamed during an appointment. However, more research needs to be conducted to confirm these hypotheses.

Date Created
2023-05
Agent

Appearance-Based Shame In Medicine: How It Alters Patient Perception and Future Care

Description

Health service quality is understood to be a crucial determinant in successful patient-physician encounters and patient health. One common feeling that patients have reported experiencing during appointments is shame. We hypothesized that patients who experience appearance-based shame during an appointment…

Health service quality is understood to be a crucial determinant in successful patient-physician encounters and patient health. One common feeling that patients have reported experiencing during appointments is shame. We hypothesized that patients who experience appearance-based shame during an appointment are not likely to return to the same physician and that patients who do not experience appearance-based shame are likely to return to the same physician. This was assessed by conducting an anonymous online survey of 13 questions that served to establish a general foundation for understanding the participants' physical characteristics such as race, age, weight, and gender identity as well as their overall patient-physician relationship and experiences of shame, if applicable. 119 participants were recruited from Arizona State University and a case study was performed individually for five participants of interest. The data analyzed from this study suggests that while appearance-based shame does exist in healthcare spaces, it is not a significant determining factor in patients returning to their physicians. In addition, there was no significant evidence to suggest that patients who do not experience appearance-based shame are either likely or more likely to return to their physician. We hypothesize this could be due to confounding variables such as convenience, accessibility, or insurance limitations which patients may prioritize over feeling ashamed during an appointment. However, more research needs to be conducted to confirm these hypotheses.

Date Created
2023-05
Agent

Dissecting the Role of Type I Proton Pyrophosphatase in Arabidopsis thaliana

Description
The partitioning of photosynthates between their sites of production (source) and their sites of utilization (sink) is a major determinant of crop yield and the potential of regulating this translocation promises substantial opportunities for yield increases. Ubiquitous overexpression of the…

The partitioning of photosynthates between their sites of production (source) and their sites of utilization (sink) is a major determinant of crop yield and the potential of regulating this translocation promises substantial opportunities for yield increases. Ubiquitous overexpression of the plant type I proton pyrophosphatase (H+-PPase) in crops improves several valuable traits including salt tolerance and drought resistance, nutrient and water use efficiencies, and increased root biomass and yield. Originally, type I H+-PPases were described as pyrophosphate (PPi)-dependent proton pumps localized exclusively in vacuoles of mesophyll and meristematic tissues. It has been proposed that in the meristematic tissues, the role of this enzyme would be hydrolyzing PPi originated in biosynthetic reactions and favoring sink strength. Interestingly, this enzyme has been also localized at the plasma membrane of companion cells in the phloem which load and transport photosynthates from source leaves to sinks. Of note, the plasma membrane-localized H+-PPase could only function as a PPi-synthase in these cells due to the steep proton gradient between the apoplast and cytosol. The generated PPi would favor active sucrose loading through the sucrose/proton symporter in the phloem by promoting sucrose hydrolysis through the Sucrose Synthase pathway and providing the ATP required to maintain the proton gradient. To better understand these two different roles of type I H+-PPases, a series of Arabidopsis thaliana transgenic plants were generated. By expressing soluble pyrophosphatases in companion cells of Col-0 ecotype and H+-PPase mutants, impaired photosynthates partitioning was observed, suggesting phloem-localized H+-PPase could generate the PPi required for sucrose loading. Col-0 plants expressed with either phloem- or meristem-specific AVP1 overexpression cassette and the cross between the two tissue specific lines (Cross) were generated. The results showed that the phloem-specific AVP1-overexpressing plants had increased root hair elongation under limited nutrient conditions and both phloem- and meristem-overexpression of AVP1 contributed to improved rhizosphere acidification and drought resistance. It was concluded that H+-PPases localized in both sink and source tissues regulate plant growth and performance under stress through its versatile enzymatic functions (PPi hydrolase and synthase).
Date Created
2022
Agent

Cellular and Molecular Mechanisms of Macrophage Fusion

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Description
Macrophage fusion resulting multinucleated giant cells (MGCs) formation is associated with numerous chronic inflammatory diseases including the foreign body reaction to implanted biomaterials. Despite long-standing predictions, there have been attempts to use live-cell imaging to investigate the morphological features initiating…

Macrophage fusion resulting multinucleated giant cells (MGCs) formation is associated with numerous chronic inflammatory diseases including the foreign body reaction to implanted biomaterials. Despite long-standing predictions, there have been attempts to use live-cell imaging to investigate the morphological features initiating macrophage fusion because macrophages do not fuse on clean glass required for most imaging techniques. Consequently, the mechanisms of macrophage fusion remain poorly understood. The goal of this research project was to characterize the early and late stages of macrophage multinucleation using fusogenic optical quality substrate. Live-cell imaging with phase-contrast and lattice-light sheet microscopy revealed that an actin-based protrusion initiates macrophage fusion. WASpdeficient macrophages and macrophages isolated from myeloid cell-specific Cdc42-/- mice fused at very low rates. In addition, inhibiting the Arp2/3 complex impaired both the formation of podosomes and macrophage fusion. Analyses of the late stages of macrophage multinucleation on biomaterials implanted into mice revealed novel actin-based zipper-like structures (ZLSs) formed at contact sites between MGCs. The model system that was developed for the induction of ZLSs in vitro allowed for the characterization of protein composition using confocal and super-resolution microscopy. Live-cell imaging demonstrated that ZLSs are dynamic formations undergoing continuous assembly and disassembly and that podosomes are precursors of these structures. It was further found that E-cadherin and nectin-2 are involved in ZLS formation by bridging the plasma membranes together. ii Macrophage fusion on implanted biomaterials inherently involves their adhesion to the implant surface. While biomaterials rapidly acquire a layer of host proteins, a biological substrate that is required for macrophage fusion is unknown. It was shown that mice with fibrinogen deficiency as well as mice expressing fibrinogen incapable of fibrin polymerization displayed a dramatic reduction of macrophage fusion on biomaterials. Furthermore, these mice were protected from the formation of the dense collagenous capsule enveloping the implant. It was also found that the main cell type responsible for the deposition of collagen in the capsule were mononuclear macrophages but not myofibroblasts. Together, these findings reveal a critical role of the actin cytoskeleton in macrophage fusion and identify potential targets to reduce the drawbacks of macrophage fusion on implanted biomaterials.
Date Created
2021
Agent

Ceramic Sculpture Exploring the Debate Surrounding Animal Testing

Description

There is a wide intersection where animal and human lives interact or mimic each other behaviorally or biologically. A lot of the products that are part of our day-to-day were first validated by animals, and eventually found their way to…

There is a wide intersection where animal and human lives interact or mimic each other behaviorally or biologically. A lot of the products that are part of our day-to-day were first validated by animals, and eventually found their way to us. From food to beauty products to scientific developments, animals deal with a lot behind the scenes. Some humans are cognizant of what is happening backstage, while others only see the final presentation. Either way, all of us have our opinions in support or against animal treatment. The project is heavily inspired from my experience in a neurorehabilitation lab, so the foundation is similar to the structure and function of neurons. Through this project, I am focusing on one aspect of this debate, which is animal testing in the scietific setting. The goal of the project is not to force the viewer to choose one side, but to understand the big picture and the reasoning of the opposing side.

Date Created
2021-05
Agent
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