Matching Items (146)
Description
In order to determine whether the spatial organization of FRCs and their expression of maturation markers (such as Ltbr) are altered with age, I performed immunofluorescence on frozen and cryosectioned whole lymph nodes from young and aged mice. My second aim was to perform RT-qPCR and flow cytometry in order to determine whether FRCs from aged mice have altered expression of maturation markers when compared to young mice. Thus, the goal of the honors thesis research was to determine whether lymph node FRCs in the aged mouse exhibit signs of impaired maturation in their protein and gene expression. As the immune system is profoundly impacted by aging, my project supports a cellular mechanism by which defects in aged tissues disrupt immune cell function. Therefore, understanding the age-associated decline in host defense could provide new avenues for the treatment of many diseases of which the elderly are most vulnerable, in particular re-emerging and novel pathological agents such as COVID-19.
ContributorsMorris, Karina (Author) / Lake, Douglas (Thesis director) / Lancaster, Jessica (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Microfluidic platforms have been exploited extensively as a tool for the separation of particles by electric field manipulation. Microfluidic devices can facilitate the manipulation of particles by dielectrophoresis. Separation of particles by size and type has been demonstrated by insulator-based dielectrophoresis in a microfluidic device. Thus, manipulating particles by size has been widely studied throughout the years. It has been shown that size-heterogeneity in organelles has been linked to multiple diseases from abnormal organelle size. Here, a mixture of two sizes of polystyrene beads (0.28 and 0.87 μm) was separated by a ratchet migration mechanism under a continuous flow (20 nL/min). Furthermore, to achieve high-throughput separation, different ratchet devices were designed to achieve high-volume separation. Recently, enormous efforts have been made to manipulate small size DNA and proteins. Here, a microfluidic device comprising of multiple valves acting as insulating constrictions when a potential is applied is presented. The tunability of the electric field gradient is evaluated by a COMSOL model, indicating that high electric field gradients can be reached by deflecting the valve at a certain distance. Experimentally, the tunability of the dynamic constriction was demonstrated by conducting a pressure study to estimate the gap distance between the valve and the substrate at different applied pressures. Finally, as a proof of principle, 0.87 μm polystyrene beads were manipulated by dielectrophoresis. These microfluidic platforms will aid in the understanding of size-heterogeneity of organelles for biomolecular assessment and achieve separation of nanometer-size DNA and proteins by dielectrophoresis.
ContributorsOrtiz, Ricardo (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2021
Description
In cold chain tracking systems, accuracy and flexibility across different temperatures ranges plays an integral role in monitoring biospecimen integrity. However, while two common cold chain tracking systems are currently available (electronic and physics/chemical), there is not an affordable cold chain tracking mechanism that can be applied to a variety of temperatures while maintaining accuracy for individual vials. Hence, our lab implemented our understanding of biochemical reaction kinetics to develop a new cold chain tracking mechanism using the permanganate/oxalic acid reaction. The permanganate/oxalic acid reaction is characterized by the reduction of permanganate (MnVII) to Mn(II) with Mn(II)-autocatalyzed oxidation of oxalate to CO2, resulting in a pink to colorless visual indicator change when the reaction system is not in the solid state (i.e., frozen or vitrified). Throughout our research, we demonstrate, (i) Improved reaction consistency and accuracy along with extended run times with the implementation of a nitric acid-based labware washing protocol, (ii) Simulated reaction kinetics for the maximum length reaction and 60-minute reaction based on previously developed MATLAB scripts (iii) Experimental reaction kinetics to verify the simulated MATLAB maximum and 60-minute reactions times (iv) Long-term stability of the permanganate/oxalic acid reaction with water or eutectic solutions of sodium perchlorate and magnesium perchlorate at -80°C (v) Reaction kinetics with eutectic solvents, sodium perchlorate and magnesium perchlorate, at 25°C, 4°C, and -8°C (vi) Accelerated reaction kinetics after the addition of varying concentrations of manganese perchlorate (vii) Reaction kinetics of higher concentration reaction systems (5x and 10x; for darker colors), at 25°C (viii) Long-term stability of the 10x higher concentration reaction at -80°C.
ContributorsLjungberg, Emil (Author) / Borges, Chad (Thesis director) / Levitus, Marcia (Committee member) / Williams, Peter (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor) / Department of Psychology (Contributor)
Created2022-12
Description
Plasma and serum are the most commonly used liquid biospecimens in biomarker research. These samples may be subjected to several pre-analytical variables (PAVs) during collection, processing and storage. Exposure to thawed conditions (temperatures above -30 °C) is a PAV that is hard to control, and track and could provide misleading information, that fail to accurately reveal the in vivo biological reality, when unaccounted for. Hence, assays that can empirically check the integrity of plasma and serum samples are crucial. As a solution to this issue, an assay titled ΔS-Cys-Albumin was developed and validated. The reference range of ΔS-Cys-Albumin in cardio vascular patients was determined and the change in ΔS-Cys-Albumin values in different samples over time course incubations at room temperature, 4 °C and -20 °C were evaluated. In blind challenges, this assay proved to be successful in identifying improperly stored samples individually and as groups. Then, the correlation between the instability of several clinically important proteins in plasma from healthy and cancer patients at room temperature, 4 °C and -20 °C was assessed. Results showed a linear inverse relationship between the percentage of proteins destabilized and ΔS-Cys-Albumin regardless of the specific time or temperature of exposure, proving ΔS-Cys-Albumin as an effective surrogate marker to track the stability of clinically relevant analytes in plasma. The stability of oxidized LDL in serum at different temperatures was assessed in serum samples and it stayed stable at all temperatures evaluated. The ΔS-Cys-Albumin requires the use of an LC-ESI-MS instrument which limits its availability to most clinical research laboratories. To overcome this hurdle, an absorbance-based assay that can be measured using a plate reader was developed as an alternative to the ΔS-Cys-Albumin assay. Assay development and analytical validation procedures are reported herein. After that, the range of absorbance in plasma and serum from control and cancer patients were determined and the change in absorbance over a time course incubation at room temperature, 4 °C and -20 °C was assessed. The results showed that the absorbance assay would act as a good alternative to the ΔS-Cys-Albumin assay.
ContributorsJehanathan, Nilojan (Author) / Borges, Chad (Thesis advisor) / Guo, Jia (Committee member) / Van Horn, Wade (Committee member) / Arizona State University (Publisher)
Created2022
Description
IOsteosarcoma is the most common bone cancer and typically affects patients in the second decade of life. Current treatment methods have not proven effective for treating reoccurring or metastatic osteosarcoma (mOS) given the 5-year survival rate of 15-30%. Previous work showed that using the immune system to fight the cancer significantly improved survival of mOS in mice, but approximately 40-50% of treated mice still succumbed to disease. To further improve immunotherapy, I analyzed immune cells in the tumor bed and observed high numbers of a rare T cell subtype: CD4hiCD8αhi, or double positive (DP), T cells. While previous literature found mature DP T cells in chronic diseases, the associations and functions of this rare T cell subtype varied between studies and were unknown for mOS. Controlling for age, chronicity of disease, and environmental exposure, I found DP T cells composed a higher percentage of T cells in the cancer as tumor burden increased. I then tested whether the DP cells were pro- or anti-tumor. I found that DP cells produced the cytokines IFNγ and IL-2 when exhaustion was overcome. They also expressed FasL for cytotoxic function, although the target is unknown. These findings suggest DP T cells have multifunctionality, which could be advantageous when responding to high antigen load.
II
Course-based undergraduate research experiences (CUREs) offer students opportunities to engage in critical thinking and problem solving. However, quantitating the impact that incorporating research into undergraduate courses has on student learning has been difficult since most CUREs lack a comparable traditional course as a control. Because the overall class structure remained unaltered when our upper division immunology course transitioned to a CURE class, we realized retrospectively that we were in a unique position to quantitate the impact of incorporating research on student performance. I then analyzed the summative assessments used to assess student learning and found that students in the CURE format class performed significantly better on quizzes, exams, and reports. There were no significant differences in academic levels, degree programs, or grade point averages, suggesting improved performance was due to increased engagement of students in research.
ContributorsAppel, Nicole (Author) / Blattman, Joseph (Thesis advisor) / Anderson, Karen (Committee member) / Lake, Douglas (Committee member) / Hingorani, Pooja (Committee member) / Arizona State University (Publisher)
Created2022
Description
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) that emerged from a zoonotic host at the end of 2019 and caused a public health crisis. In this collection of studies, Nicotiana benthamiana plants are used to set the foundation for producing monoclonal antibodies (mAbs) with homogeneous glycosylation to neutralize SARS-CoV-2 and potentially address the immunopathology often observed with severe COVID-19. Specifically, a mAb against the human interleukin (IL)-6 receptor (sarilumab) was generated and evaluated in vitro for its potential to reduce IL-6 signaling that has been shown to be associated with more severe cases of COVID-19. Furthermore, multiple mAbs that bind to the receptor-binding domain (RBD) of SARS-CoV-2 and efficiently neutralize the virus were developed using plant-based expression. Several of these mAbs are from different classes of RBD-binding mAbs that have distinct binding sites from one another. Several mAbs from different classes showed synergy in neutralizing the ancestral strain of SARS-CoV-2 and a smaller subset showed synergy when tested against the highly mutated Omicron (B.1.1.529) variant. Of interest, a novel RBD-binding mAb, termed 11D7, that was raised against the ancestral strain and derived from a hybridoma, appears to have an epitope on the RBD that contributes more synergy to a mAb combination that efficiently neutralizes the B.1.1.529 variant of SARS-CoV-2. This epitope was partially mapped by competitive binding and shows that it overlaps with another known antibody that binds a cryptic, distal epitope, away from the receptor binding site, giving insight into the potential mechanism by which 11D7 neutralizes SARS-CoV-2, as well as potentially allowing it to resist SARS-CoV-2 immune evasion more efficiently. Furthermore, this mAb carries a highly homogeneous glycan pattern when expressed in N. benthamiana, that may contribute to enhanced effector function and provides a tool to elucidate the precise role of crystallizable fragment (Fc)-mediated protection in SARS-CoV-2 infection. Ultimately, these studies provide evidence of the utility of plant-made mAbs to be used as cocktail members, giving clarity to the use of less potent mAbs as valuable cocktail components which will spur further investigations into how mAbs with unique epitopes work together to efficiently neutralize SARS-CoV-2.
ContributorsJugler, Collin (Author) / Chen, Qiang (Thesis advisor) / Lake, Douglas (Committee member) / Steele, Kelly (Committee member) / Mason, Hugh (Committee member) / Arizona State University (Publisher)
Created2022
Description
The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic, declared in March 2020 resulted in an unprecedented scientific effort that led to the deployment in less than a year of several vaccines to prevent severe disease, hospitalizations, and death from coronavirus disease 2019 (COVID-19). Most vaccine models focus on the production of neutralizing antibodies against the spike (S) to prevent infection. As the virus evolves, new variants emerge that evade neutralizing antibodies produced by natural infection and vaccination, while memory T cell responses are long-lasting and resilient to most of the changes found in variants of concern (VOC). Several lines of evidence support the study of T cell-mediated immunity in SARS-CoV-2 infections. First, T cell reactivity against SARS-CoV-2 is found in both (cluster of differentiation) CD4+ and CD8+ T cell compartments in asymptomatic, mild, and severe recovered COVID-19 patients. Second, an early and stronger CD8+ T cell response correlates with less severe COVID-19 disease [1-4]. Third, both CD4+ and CD8+ T cells that are reactive to SARS-CoV-2 viral antigens are found in healthy unexposed individuals suggesting that cross-reactive and conserved epitopes may be protective against infection.
The current study is focused on the T cell-mediated response, with special attention to conserved, non-spike-cross-reactive epitopes that may be protective against SARS-CoV-2. The first chapter reviews the importance of epitope prediction in understanding the T cell-mediated responses to a pathogen. The second chapter centers on the validation of SARS-CoV-2 CD8+ T cell predicted peptides to find conserved, immunodominant, and immunoprevalent epitopes that can be incorporated into the next generation of vaccines against severe COVID-19 disease. The third chapter explores pre-existing immunity to SARS-CoV-2 in a pre-pandemic cohort and finds two highly immunogenic epitopes that are conserved among human common cold coronaviruses (HCoVs). To end, the fourth chapter explores the concept of T cell receptor (TCR) cross-reactivity by isolating SARS-CoV-2-reactive TCRs to elucidate the mechanisms of cross-reactivity to SARS-CoV-2 and other human coronaviruses (HCoVs).
ContributorsCarmona, Jacqueline (Author) / Anderson, Karen S (Thesis advisor) / Lake, Douglas (Thesis advisor) / Maley, Carlo (Committee member) / Mangone, Marco (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2023
Description
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative pathogen of Coronavirus Disease 2019 (COVID-19). Successful vaccination aims to elicit neutralizing antibodies (NAbs) which inhibit viral infection. Traditional NAb quantification methods (neutralization assays) are labor-intensive and expensive, with limited practicality for routine use (e.g. monitoring vaccination response). Thus, a rapid (10-minute) lateral flow assay (LFA) for quantification of SARS-CoV-2 NAbs was developed. Using the NAb LFA, an 18-month longitudinal study assessing monthly NAb titers was conducted in a cohort of over 500 COVID-19 mRNA vaccine recipients. Three NAb response groups were identified: vaccine strong responders (VSRs), moderate responders (VMRs), and poor responders (VPRs). VSRs generated high and durable NAb titers. VMRs initially generated high NAb titers but showed more rapid waning with time post-vaccination. Finally, VPRs rarely generated NAb titers ≥1:160, even after 3rd dose. Although strong humoral responses correlate with vaccine effectiveness, viral-specific CD4+ and CD8+ T cells are critical for long-term protection. Discordant phenotypes of viral-specific CD8+ and CD4+CXCR5+ T follicular helper (cTfh) cells have recently been associated with differential NAb responses. The second portion of this dissertation was to investigate whether/how SARS-CoV-2 T cell responses differ in individuals with impaired NAb titers following mRNA vaccination. Thus, phenotypic and functional characterization of T cell activation across NAb response groups was conducted. It was hypothesized that VPRs would exhibit discordant SARS-CoV-2 T cell activation and altered cTfh phenotypes. Peripheral blood mononuclear cells were isolated from VPRs, VMRs, VSRs, naturally infected, and normal donors. SARS-CoV-2 responsive T cells were characterized using in vitro activation induced marker assays, multicolor flow cytometry, and multiplex cytokine analysis. Further, CXCR5+ cTfh were examined for chemokine receptor expression (CCR6 and CXCR3). Results demonstrated that despite differential NAb responses, activation of SARS-CoV-2 responsive CD4+ and CD8+ T cells was comparable across NAb groups. However, double-positive CD4+CD8+, CD8low, and activated CD4+CXCR5+CCR6-CXCR3+ (Tfh1-like) T cells were expanded in VPRs compared to VMR and VSRs. Interestingly, a unique population of CD8+CXCR5+ T cells was also expanded in VPRs. These novel findings may aid in identification of individuals with impaired or altered immune responses to COVID-19 mRNA vaccination.
ContributorsRoeder, Alexa Jordan (Author) / Lake, Douglas (Thesis advisor) / McFadden, Grant (Committee member) / Borges Florsheim, Esther (Committee member) / Chang, Yung (Committee member) / Rahman, Masmudur (Committee member) / Arizona State University (Publisher)
Created2023
Description
Trace evidence is an essential component of forensic investigations. Anthropogenicmaterials such as fibers and glass have been well studied for use in forensic trace evidence, but the potential use of retroreflective beads found in soils for forensic investigations is largely unexplored. Retroreflective glass beads are tiny spheres mixed into pavement markings to create reflective surfaces to reduce lane departure accidents. Retroreflective glass beads are a potentially new source of trace evidence for forensic investigations.
Analysis of the spatial distribution and chemical compositions of retroreflective glass beads recovered from 17 soil samples were analyzed and compared to see if there are striking variations that can distinguish samples by source. Soil samples taken near marked roads showed significantly higher concentrations of glass beads, averaging from 0.18 bead/g of soil sample to 587 beads/g of soil, while soil samples taken near unmarked roads had average range of concentration of 0 bead/g of soil to 0.21 bead/g of soil. Retroreflective glass beads come from pavement markings, thus soil samples near marked roads are expected to have higher concentrations of glass beads. Analysis of spatial distribution of glass beads showed that as sample collection moved further from the road, concentration of glass beads decreased.
ICP-MS results of elemental concentrations for each sample showed discriminative differences between samples, for most of the elements. An analysis of variance for elemental concentrations was conducted, and results showed statistically significant differences, beyond random chance alone for half of the elements analyzed. For forensic comparisons, a significant difference in even just one element is enough to conclude that the samples came from different sources. The elemental concentrations of glass beads collected from the same location, but of varying differences, was also analyzed. ANOVA results show significant differences for only one or two elements. A pair-wise t-test was conducted to determine which elements are most discriminative among all the samples. Rubidium was found to be the most discriminative, showing significant difference for 67% of the pairs. Beryllium, potassium, and manganese were also highly discriminative, showing significant difference for at least 50% of all the pairs.
ContributorsGomez, Janelle Kate Pacifico (Author) / Montero, Shirly (Thesis advisor) / Herckes, Pierre (Thesis advisor) / Borges, Chad (Committee member) / Gordon, Gwyneth (Committee member) / Arizona State University (Publisher)
Created2023
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