Matching Items (27)
Filtering by
- Genre: Doctoral Dissertation
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
Type 1 diabetes (T1D) is the result of an autoimmune attack against the insulin-producing β-cells of the pancreas causing hyperglycemia and requiring the individual to rely on life-long exogenous insulin. With the age of onset typically occurring in childhood, there is increased physical and emotional stress to the child as well as caregivers to maintain appropriate glucose levels. The majority of T1D patients have antibodies to one or more antigens: insulin, IA-2, GAD65, and ZnT8. Although antibodies are detectable years before symptoms occur, the initiating factors and mechanisms of progression towards β-cell destruction are still not known. The search for new autoantibodies to elucidate the autoimmune process in diabetes has been slow, with proteome level screenings on native proteins only finding a few minor antigens. Post-translational modifications (PTM)—chemical changes that occur to the protein after translation is complete—are an unexplored way a self-protein could become immunogenic. This dissertation presents the first large sale screening of autoantibodies in T1D to nitrated proteins. The Contra Capture Protein Array (CCPA) allowed for fresh expression of hundreds of proteins that were captured on a secondary slide by tag-specific ligand and subsequent modification with peroxynitrite. The IgG and IgM humoral response of 48 newly diagnosed T1D subjects and 48 age-matched controls were screened against 1632 proteins highly or specifically expressed in pancreatic cells. Top targets at 95% specificity were confirmed with the same serum samples using rapid antigenic protein in situ display enzyme-linked immunosorbent assay (RAPID ELISA) a modified sandwich ELISA employing the same cell-free expression as the CCPA. For validation, 8 IgG and 5 IgM targets were evaluated with an independent serum sample set of 94 T1D subjects and 94 controls. The two best candidates at 90% specificity were estrogen receptor 1 (ESR1) and phosphatidylinositol 4-kinase type 2 beta (PI4K2B) which had sensitivities of 22% (p=.014) and 25% (p=.045), respectively. Receiver operating characteristic (ROC) analyses found an area under curve (AUC) of 0.6 for ESR1 and 0.58 for PI4K2B. These studies demonstrate the ability and value for high-throughput autoantibody screening to modified antigens and the frequency of Type 1 diabetes.
ContributorsHesterman, Jennifer (Author) / LaBaer, Joshua (Thesis advisor) / Borges, Chad (Committee member) / Sweazea, Karen (Committee member) / Mangone, Marco (Committee member) / Arizona State University (Publisher)
Created2022
Description
Antibodies are the immunoglobulins which are secreted by the B cells after a microbial invasion. They are stable and stays in the serum for a long time which makes them an excellent biomarker for disease diagnosis. Inflammatory bowel disease is a type of autoimmune disease where the immune system mistakenly attacks the commensal bacteria and leads to inflammation. We studied antibody response of 100 Crohn’s disease (CD), 100 ulcerative colitis (UC) and 100 healthy controls against 1,173 bacterial and 397 viral proteins. We found some anti-bacterial antibodies higher in CD compared to controls while some antibodies lower in UC compared to controls. We were able to build biomarker panels with AUCs of 0.81, 0.87, and 0.82 distinguishing CD vs. control, UC vs. control, and CD vs. UC, respectively. Subgroup analysis based on the Montreal classification revealed that penetrating CD behavior (B3), colonic CD location (L2), and extensive UC (E3) exhibited highest antibody reactivity among all patients. We also wanted to study the reason for the presence of autoantibodies in the sera of healthy individuals. A meta-analysis of 9 independent biomarker study was performed to find 77 common autoantibodies shared by healthy individuals. There was no gender bias; however, the number of autoantibodies increased with age, plateauing around adolescence. Molecular mimicry likely contributed to the elicitation of a subset of these common autoantibodies as 21 common autoantigens had 7 or more ungapped amino acid matches with viral proteins. Intrinsic properties of protein like hydrophilicity, basicity, aromaticity, and flexibility were enriched for common autoantigens. Subcellular localization and tissue expression analysis indicated the sequestration of some autoantigens from circulating autoantibodies can explain the absence of autoimmunity in these healthy individuals.
ContributorsShome, Mahasish (Author) / LaBaer, Joshua (Thesis advisor) / Borges, Chad (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2021
Description
Transient protein-protein and protein-molecule interactions fluctuate between associated and dissociated states. They are widespread in nature and mediate most biological processes. These interactions are complex and are strongly influenced by factors such as concentration, structure, and environment. Understanding and utilizing these types of interactions is useful from both a fundamental and design perspective. In this dissertation, transient protein interactions are used as the sensing element of a biosensor for small molecule detection. This is done by using a transcription factor-small molecule pair that mediates the activation of a CRISPR/Cas12a complex. Activation of the Cas12a enzyme results in an amplified readout mechanism that is either fluorescence or paper based. This biosensor can successfully detect 9 different small molecules including antibiotics with a tuneable detection limit ranging from low µM to low nM. By combining protein and nucleic acid-based systems, this biosensor has the potential to report on almost any protein-molecule interaction, linking this to the intrinsic amplification that is possible when working with nucleic acid-based technologies. The second part of this dissertation focuses on understanding protein-molecule interactions at a more fundamental level, and, in so doing, exploring design rules required to generalize sensors like the ones described above. This is done by training a neural network algorithm with binding data from high density peptide micro arrays incubated with specific protein targets. Because the peptide sequences were chosen simply to evenly, though sparsely, represent all sequence space, the resulting network provides a comprehensive sequence/binding relationship for a given target protein. While past work had shown that this works well on the arrays, here I have explored how well the neural networks thus trained, predict sequence-dependent binding in the context of protein-protein and peptide-protein interactions. Amino acid sequences, either free in solution or embedded in protein structure, will display somewhat different binding properties than sequences affixed to the surface of a high-density array. However, the neural network trained on array sequences was able to both identify binding regions in between proteins and predict surface plasmon resonance-based binding propensities for peptides with statistically significant levels of accuracy.
ContributorsSwingle, Kirstie Lynn (Author) / Woodbury, Neal W (Thesis advisor) / Green, Alexander A (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2022
Description
Tools designed to help match people with behaviors they identify as likely to lead to a successful behavioral outcome remain under-researched. This study assessed the effect of a participant-driven behavior-matching intervention on 1) the adoption of a new behavior related to fruit and vegetable (F&V) consumption, 2) study attrition, and 3) changes in F&V consumption. In this two-arm randomized controlled trial, 64 adults who did not meet standard F&V recommendations were allocated to an intervention (n=33) or control group (n=31). Participants in the intervention group ranked 20 F&V-related behaviors according to their perceived likelihood of engagement in the behavior and their perception of the behavior’s efficacy in increasing F&V consumption. Participants in the intervention group were subsequently shown the list of 20 behaviors in order of their provided rankings, with the highest-ranked behaviors at the top, and were asked to choose a behavior they would like to perform daily for 4 weeks. The control group chose from a random-order list of the same 20 behaviors to adopt daily for 4 weeks. During the study period, text messages were sent to all participants 90 minutes before their reported bedtime to collect Yes/No data reflecting successful behavior engagement each day. The binary repeated-measures data collected from the text messages was analyzed using mixed-effects logistic regression, differences in attrition were assessed using log-rank analysis, and change scores in F&V consumption were compared between the two groups using the Man-Whitney U test. P<0.05 indicated significance. The rate of successful behavior adoption did not differ significantly between the two groups (b=0.09, 95%CI= -0.81, 0.98, p=0.85). The log rank test results indicated that there was no significant difference in attrition between the two groups (χ2=2.68, df=1, p=0.10). F&V consumption increased significantly over the 4 weeks in the total sample (Z=-5.86, p<0.001), but no differences in F&V change scores were identified between the control and intervention groups (Z=-0.21, p=0.84). The behavior-matching tool assessed in this study did not significantly improve behavior adoption, study attrition, or F&V intake over 4 weeks.
ContributorsCosgrove, Kelly Sarah (Author) / Wharton, Christopher (Thesis advisor) / Adams, Marc (Committee member) / DesRoches, Tyler (Committee member) / Grebitus, Carola (Committee member) / Johnston, Carol (Committee member) / Arizona State University (Publisher)
Created2023
Description
Measuring molecular interaction with membrane proteins is critical for understanding cellular functions, validating biomarkers and screening drugs. Despite the importance, developing such a capability has been a difficult challenge, especially for small molecules binding to membrane proteins in their native cellular environment. The current mainstream practice is to isolate membrane proteins from the cell membranes, which is difficult and often lead to the loss of their native structures and functions. In this thesis, novel detection methods for in situ quantification of molecular interactions with membrane proteins are described.
First, a label-free surface plasmon resonance imaging (SPRi) platform is developed for the in situ detection of the molecular interactions between membrane protein drug target and its specific antibody drug molecule on cell surface. With this method, the binding kinetics of the drug-target interaction is quantified for drug evaluation and the receptor density on the cell surface is also determined.
Second, a label-free mechanically amplification detection method coupled with a microfluidic device is developed for the detection of both large and small molecules on single cells. Using this method, four major types of transmembrane proteins, including glycoproteins, ion channels, G-protein coupled receptors (GPCRs) and tyrosine kinase receptors on single whole cells are studied with their specific drug molecules. The basic principle of this method is established by developing a thermodynamic model to express the binding-induced nanometer-scale cellular deformation in terms of membrane protein density and cellular mechanical properties. Experiments are carried out to validate the model.
Last, by tracking the cell membrane edge deformation, molecular binding induced downstream event – granule exocytosis is measured with a dual-optical imaging system. Using this method, the single granule exocytosis events in single cells are monitored and the temporal-spatial distribution of the granule fusion-induced cell membrane deformation are mapped. Different patterns of granule release are resolved, including multiple release events occurring close in time and position. The label-free cell membrane deformation tracking method was validated with the simultaneous fluorescence recording. And the simultaneous cell membrane deformation detection and fluorescence recording allow the study of the propagation of the granule release-induced membrane deformation along cell surfaces.
First, a label-free surface plasmon resonance imaging (SPRi) platform is developed for the in situ detection of the molecular interactions between membrane protein drug target and its specific antibody drug molecule on cell surface. With this method, the binding kinetics of the drug-target interaction is quantified for drug evaluation and the receptor density on the cell surface is also determined.
Second, a label-free mechanically amplification detection method coupled with a microfluidic device is developed for the detection of both large and small molecules on single cells. Using this method, four major types of transmembrane proteins, including glycoproteins, ion channels, G-protein coupled receptors (GPCRs) and tyrosine kinase receptors on single whole cells are studied with their specific drug molecules. The basic principle of this method is established by developing a thermodynamic model to express the binding-induced nanometer-scale cellular deformation in terms of membrane protein density and cellular mechanical properties. Experiments are carried out to validate the model.
Last, by tracking the cell membrane edge deformation, molecular binding induced downstream event – granule exocytosis is measured with a dual-optical imaging system. Using this method, the single granule exocytosis events in single cells are monitored and the temporal-spatial distribution of the granule fusion-induced cell membrane deformation are mapped. Different patterns of granule release are resolved, including multiple release events occurring close in time and position. The label-free cell membrane deformation tracking method was validated with the simultaneous fluorescence recording. And the simultaneous cell membrane deformation detection and fluorescence recording allow the study of the propagation of the granule release-induced membrane deformation along cell surfaces.
ContributorsZhang, Fenni (Author) / Tao, Nongjian (Thesis advisor) / Chae, Junseok (Committee member) / Borges, Chad (Committee member) / Jing, Tianwei (Committee member) / Wang, Shaopeng (Committee member) / Arizona State University (Publisher)
Created2018
Description
Measurements of different molecular species from single cells have the potential to reveal cell-to-cell variations, which are precluded by population-based measurements. An increasing percentage of researches have been focused on proteins, for its central roles in biological processes. Immunofluorescence (IF) has been a well-established protein analysis platform. To gain comprehensive insights into cell biology and diagnostic pathology, a crucial direction would be to increase the multiplexity of current single cell protein analysis technologies.
An azide-based chemical cleavable linker has been introduced to design and synthesis novel fluorescent probes. These probes allow cyclic immunofluorescence staining which leads to the feasibility of highly multiplexed single cell in situ protein profiling. These highly multiplexed imaging-based platforms have the potential to quantify more than 100 protein targets in cultured cells and more than 50 protein targets in single cells in tissues.
This approach has been successfully applied in formalin-fixed paraffin-embedded (FFPE) brain tissues. Multiplexed protein expression level results reveal neuronal heterogeneity in the human hippocampus.
An azide-based chemical cleavable linker has been introduced to design and synthesis novel fluorescent probes. These probes allow cyclic immunofluorescence staining which leads to the feasibility of highly multiplexed single cell in situ protein profiling. These highly multiplexed imaging-based platforms have the potential to quantify more than 100 protein targets in cultured cells and more than 50 protein targets in single cells in tissues.
This approach has been successfully applied in formalin-fixed paraffin-embedded (FFPE) brain tissues. Multiplexed protein expression level results reveal neuronal heterogeneity in the human hippocampus.
ContributorsLiao, Renjie (Author) / Guo, Jia (Thesis advisor) / Borges, Chad (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2019
Description
DNA and RNA are generally regarded as one of the central molecules in molecular biology. Recent advancements in the field of DNA/RNA nanotechnology witnessed the success of usage of DNA/RNA as programmable molecules to construct nano-objects with predefined shapes and dynamic molecular machines for various functions. From the perspective of structural design with nucleic acid, there are basically two types of assembly method, DNA tile based assembly and DNA origami based assembly, used to construct infinite-sized crystal structures and finite-sized molecular structures. The assembled structure can be used for arrangement of other molecules or nanoparticles with the resolution of nanometers to create new type of materials. The dynamic nucleic acid machine is based on the DNA strand displacement, which allows two nucleic acid strands to hybridize with each other to displace one or more prehybridized strands in the process. Strand displacement reaction has been implemented to construct a variety of dynamic molecular systems, such as molecular computer, oscillators, in vivo devices for gene expression control.
This thesis will focus on the computational design of structural and dynamic nucleic acid systems, particularly for new type of DNA structure design and high precision control of gene expression in vivo. Firstly, a new type of fundamental DNA structural motif, the layered-crossover motif, will be introduced. The layered-crossover allow non-parallel alignment of DNA helices with precisely controlled angle. By using the layered-crossover motif, the scaffold can go through the 3D framework DNA origami structures. The properties of precise angle control of the layered-crossover tiles can also be used to assemble 2D and 3D crystals. One the dynamic control part, a de-novo-designed riboregulator is developed that can recognize single nucleotide variation. The riboregulators can also be used to develop paper-based diagnostic devices.
This thesis will focus on the computational design of structural and dynamic nucleic acid systems, particularly for new type of DNA structure design and high precision control of gene expression in vivo. Firstly, a new type of fundamental DNA structural motif, the layered-crossover motif, will be introduced. The layered-crossover allow non-parallel alignment of DNA helices with precisely controlled angle. By using the layered-crossover motif, the scaffold can go through the 3D framework DNA origami structures. The properties of precise angle control of the layered-crossover tiles can also be used to assemble 2D and 3D crystals. One the dynamic control part, a de-novo-designed riboregulator is developed that can recognize single nucleotide variation. The riboregulators can also be used to develop paper-based diagnostic devices.
ContributorsHong, Fan, Ph. D (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Green, Alexander A. (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2019
Description
Dielectrophoresis (DEP) is a technique that influences the motion of polarizable particles in an electric field gradient. DEP can be combined with other effects that influence the motion of a particle in a microchannel, such as electrophoresis and electroosmosis. Together, these three can be used to probe properties of an analyte, including charge, conductivity, and zeta potential. DEP shows promise as a high-resolution differentiation and separation method, with the ability to distinguish between subtly-different populations. This, combined with the fast (on the order of minutes) analysis times offered by the technique, lend it many of the features necessary to be used in rapid diagnostics and point-of-care devices.
Here, a mathematical model of dielectrophoretic data is presented to connect analyte properties with data features, including the intercept and slope, enabling DEP to be used in applications which require this information. The promise of DEP to distinguish between analytes with small differences is illustrated with antibiotic resistant bacteria. The DEP system is shown to differentiate between methicillin-resistant and susceptible Staphylococcus aureus. This differentiation was achieved both label free and with bacteria that had been fluorescently-labeled. Klebsiella pneumoniae carbapenemase-positive and negative Klebsiella pneumoniae were also distinguished, demonstrating the differentiation for a different mechanism of antibiotic resistance. Differences in dielectrophoretic behavior as displayed by S. aureus and K. pneumoniae were also shown by Staphylococcus epidermidis. These differences were exploited for a separation in space of gentamicin-resistant and -susceptible S. epidermidis. Besides establishing the ability of DEP to distinguish between populations with small biophysical differences, these studies illustrate the possibility for the use of DEP in applications such as rapid diagnostics.
Here, a mathematical model of dielectrophoretic data is presented to connect analyte properties with data features, including the intercept and slope, enabling DEP to be used in applications which require this information. The promise of DEP to distinguish between analytes with small differences is illustrated with antibiotic resistant bacteria. The DEP system is shown to differentiate between methicillin-resistant and susceptible Staphylococcus aureus. This differentiation was achieved both label free and with bacteria that had been fluorescently-labeled. Klebsiella pneumoniae carbapenemase-positive and negative Klebsiella pneumoniae were also distinguished, demonstrating the differentiation for a different mechanism of antibiotic resistance. Differences in dielectrophoretic behavior as displayed by S. aureus and K. pneumoniae were also shown by Staphylococcus epidermidis. These differences were exploited for a separation in space of gentamicin-resistant and -susceptible S. epidermidis. Besides establishing the ability of DEP to distinguish between populations with small biophysical differences, these studies illustrate the possibility for the use of DEP in applications such as rapid diagnostics.
ContributorsHilton, Shannon (Author) / Hayes, Mark A. (Thesis advisor) / Borges, Chad (Committee member) / Herckes, Pierre (Committee member) / Arizona State University (Publisher)
Created2019
Description
Background In the United States (US), first-year university students typically live on campus and purchase a meal plan. In general, meal plans allow the student a set number of meals per week or semester, or unlimited meals. Understanding how students’ use their meal plan, and barriers and facilitators to meal plan use, may help decrease nutrition-related issues.
Methods First-year students’ meal plan and residence information was provided by a large, public, southwestern university for the 2015-2016 academic year. A subset of students (n=619) self-reported their food security status. Logistic generalized estimating equations (GEEs) were used to determine if meal plan purchase and use were associated with food insecurity. Linear GEEs were used to examine several potential reasons for lower meal plan use. Logistic and Linear GEEs were used to determine similarities in meal plan purchase and use for a total of 599 roommate pairs (n=1186 students), and 557 floormates.
Results Students did not use all of the meals available to them; 7% of students did not use their meal plan for an entire month. After controlling for socioeconomic factors, compared to students on unlimited meal plans, students on the cheapest meal plan were more likely to report food insecurity (OR=2.2, 95% CI=1.2, 4.1). In Fall, 26% of students on unlimited meal plans reported food insecurity. Students on the 180 meals/semester meal plan who used fewer meals were more likely to report food insecurity (OR=0.9, 95% CI=0.8, 1.0); after gender stratification this was only evident for males. Students’ meal plan use was lower if the student worked a job (β=-1.3, 95% CI=-2.3, -0.3) and higher when their roommate used their meal plan frequently (β=0.09, 99% CI=0.04, 0.14). Roommates on the same meal plan (OR=1.56, 99% CI=1.28, 1.89) were more likely to use their meals together.
Discussion This study suggests that determining why students are not using their meal plan may be key to minimizing the prevalence of food insecurity on college campuses, and that strategic roommate assignments may result in students’ using their meal plan more frequently. Students’ meal plan information provides objective insights into students’ university transition.
Methods First-year students’ meal plan and residence information was provided by a large, public, southwestern university for the 2015-2016 academic year. A subset of students (n=619) self-reported their food security status. Logistic generalized estimating equations (GEEs) were used to determine if meal plan purchase and use were associated with food insecurity. Linear GEEs were used to examine several potential reasons for lower meal plan use. Logistic and Linear GEEs were used to determine similarities in meal plan purchase and use for a total of 599 roommate pairs (n=1186 students), and 557 floormates.
Results Students did not use all of the meals available to them; 7% of students did not use their meal plan for an entire month. After controlling for socioeconomic factors, compared to students on unlimited meal plans, students on the cheapest meal plan were more likely to report food insecurity (OR=2.2, 95% CI=1.2, 4.1). In Fall, 26% of students on unlimited meal plans reported food insecurity. Students on the 180 meals/semester meal plan who used fewer meals were more likely to report food insecurity (OR=0.9, 95% CI=0.8, 1.0); after gender stratification this was only evident for males. Students’ meal plan use was lower if the student worked a job (β=-1.3, 95% CI=-2.3, -0.3) and higher when their roommate used their meal plan frequently (β=0.09, 99% CI=0.04, 0.14). Roommates on the same meal plan (OR=1.56, 99% CI=1.28, 1.89) were more likely to use their meals together.
Discussion This study suggests that determining why students are not using their meal plan may be key to minimizing the prevalence of food insecurity on college campuses, and that strategic roommate assignments may result in students’ using their meal plan more frequently. Students’ meal plan information provides objective insights into students’ university transition.
Contributorsvan Woerden, Irene (Author) / Bruening, Meg (Thesis advisor) / Hruschka, Daniel (Committee member) / Schaefer, David (Committee member) / Vega-Lopez, Sonia (Committee member) / Adams, Marc (Committee member) / Arizona State University (Publisher)
Created2019