Matching Items (183)
Description
A synbody is a newly developed protein binding peptide which can be rapidly produced by chemical methods. The advantages of the synbody producing process make it a potential human proteome binding reagent. Most of the synbodies are designed to bind to specific proteins. The peptides incorporated in a synbody are discovered with peptide microarray technology. Nevertheless, the targets for unknown synbodies can also be discovered by searching through a protein mixture. The first part of this thesis mainly focuses on the process of target searching, which was performed with immunoprecipitation assays and mass spectrometry analysis. Proteins are pulled down from the cell lysate by certain synbodies, and then these proteins are identified using mass spectrometry. After excluding non-specific bindings, the interaction between a synbody and its real target(s) can be verified with affinity measurements. As a specific example, the binding between 1-4-KCap synbody and actin was discovered. This result proved the feasibility of the mass spectrometry based method and also suggested that a high throughput synbody discovery platform for the human proteome could be developed. Besides the application of synbody development, the peptide microarray technology can also be used for immunosignatures. The composition of all types of antibodies existing in one's blood is related to an individual's health condition. A method, called immunosignaturing, has been developed for early disease diagnosis based on this principle. CIM10K microarray slides work as a platform for blood antibody detection in immunosignaturing. During the analysis of an immunosignature, the data from these slides needs to be validated by using landing light peptides. The second part of this thesis focuses on the validation of the data. A biotinylated peptide was used as a landing light on the new CIM10K slides. The data was collected in several rounds of tests and indicated that the variation among landing lights was significantly reduced by using the newly prepared biotinylated peptide compared with old peptide mixture. Several suggestions for further landing light improvement are proposed based on the results.
ContributorsSun, Minyao (Author) / Johnston, Stephen Albert (Thesis advisor) / Diehnelt, Chris Wayne (Committee member) / Stafford, Phillip (Committee member) / Arizona State University (Publisher)
Created2011
Description
Filtration for microfluidic sample-collection devices is desirable for sample selection, concentration, preprocessing, and downstream manipulation, but microfabricating the required sub-micrometer filtration structure is an elaborate process. This thesis presents a simple method to fabricate polydimethylsiloxane (PDMS) devices with an integrated membrane filter that will sample, lyse, and extract the DNA from microorganisms in aqueous environments. An off-the-shelf membrane filter disc was embedded in a PDMS layer and sequentially bound with other PDMS channel layers. No leakage was observed during filtration. This device was validated by concentrating a large amount of cyanobacterium Synechocystis in simulated sample water with consistent performance across devices. After accumulating sufficient biomass on the filter, a sequential electrochemical lysing process was performed by applying 5VDC across the filter. This device was further evaluated by delivering several samples of differing concentrations of cyanobacterium Synechocystis then quantifying the DNA using real-time PCR. Lastly, an environmental sample was run through the device and the amount of photosynthetic microorganisms present in the water was determined. The major breakthroughs in this design are low energy demand, cheap materials, simple design, straightforward fabrication, and robust performance, together enabling wide-utility of similar chip-based devices for field-deployable operations in environmental micro-biotechnology.
ContributorsLecluse, Aurelie (Author) / Meldrum, Deirdre (Thesis advisor) / Chao, Joseph (Thesis advisor) / Westerhoff, Paul (Committee member) / Arizona State University (Publisher)
Created2011
Description
A novel concept for integration of flame-assisted fuel cells (FFC) with a gas turbine is analyzed in this paper. Six different fuels (CH4, C3H8, JP-4, JP-5, JP-10(L), and H2) are investigated for the analytical model of the FFC integrated gas turbine hybrid system. As equivalence ratio increases, the efficiency of the hybrid system increases initially then decreases because the decreasing flow rate of air begins to outweigh the increasing hydrogen concentration. This occurs at an equivalence ratio of 2 for CH4. The thermodynamic cycle is analyzed using a temperature entropy diagram and a pressure volume diagram. These thermodynamic diagrams show as equivalence ratio increases, the power generated by the turbine in the hybrid setup decreases. Thermodynamic analysis was performed to verify that energy is conserved and the total chemical energy going into the system was equal to the heat rejected by the system plus the power generated by the system. Of the six fuels, the hybrid system performs best with H2 as the fuel. The electrical efficiency with H2 is predicted to be 27%, CH4 is 24%, C3H8 is 22%, JP-4 is 21%, JP-5 is 20%, and JP-10(L) is 20%. When H2 fuel is used, the overall integrated system is predicted to be 24.5% more efficient than the standard gas turbine system. The integrated system is predicted to be 23.0% more efficient with CH4, 21.9% more efficient with C3H8, 22.7% more efficient with JP-4, 21.3% more efficient with JP-5, and 20.8% more efficient with JP-10(L). The sensitivity of the model is investigated using various fuel utilizations. When CH4 fuel is used, the integrated system is predicted to be 22.7% more efficient with a fuel utilization efficiency of 90% compared to that of 30%.
ContributorsRupiper, Lauren Nicole (Author) / Milcarek, Ryan (Thesis director) / Wang, Liping (Committee member) / Mechanical and Aerospace Engineering Program (Contributor) / School for Engineering of Matter,Transport & Enrgy (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Single cell phenotypic heterogeneity studies reveal more information about the pathogenesis process than conventional bulk methods. Furthermore, investigation of the individual cellular response mechanism during rapid environmental changes can only be achieved at single cell level. By enabling the study of cellular morphology, a single cell three-dimensional (3D) imaging system can be used to diagnose fatal diseases, such as cancer, at an early stage. One proven method, CellCT, accomplishes 3D imaging by rotating a single cell around a fixed axis. However, some existing cell rotating mechanisms require either intricate microfabrication, and some fail to provide a suitable environment for living cells. This thesis develops a microvorterx chamber that allows living cells to be rotated by hydrodynamic alone while facilitating imaging access. In this thesis work, 1) the new chamber design was developed through numerical simulation. Simulations revealed that in order to form a microvortex in the side chamber, the ratio of the chamber opening to the channel width must be smaller than one. After comparing different chamber designs, the trapezoidal side chamber was selected because it demonstrated controllable circulation and met the imaging requirements. Microvortex properties were not sensitive to the chambers with interface angles ranging from 0.32 to 0.64. A similar trend was observed when chamber heights were larger than chamber opening. 2) Micro-particle image velocimetry was used to characterize microvortices and validate simulation results. Agreement between experimentation and simulation confirmed that numerical simulation was an effective method for chamber design. 3) Finally, cell rotation experiments were performed in the trapezoidal side chamber. The experimental results demonstrated cell rotational rates ranging from 12 to 29 rpm for regular cells. With a volumetric flow rate of 0.5 µL/s, an irregular cell rotated at a mean rate of 97 ± 3 rpm. Rotational rates can be changed by altering inlet flow rates.
ContributorsZhang, Wenjie (Author) / Frakes, David (Thesis advisor) / Meldrum, Deirdre (Thesis advisor) / Chao, Shih-hui (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2011
Description
The technology expansion seen in the last decade for genomics research has permitted the generation of large-scale data sources pertaining to molecular biological assays, genomics, proteomics, transcriptomics and other modern omics catalogs. New methods to analyze, integrate and visualize these data types are essential to unveil relevant disease mechanisms. Towards these objectives, this research focuses on data integration within two scenarios: (1) transcriptomic, proteomic and functional information and (2) real-time sensor-based measurements motivated by single-cell technology. To assess relationships between protein abundance, transcriptomic and functional data, a nonlinear model was explored at static and temporal levels. The successful integration of these heterogeneous data sources through the stochastic gradient boosted tree approach and its improved predictability are some highlights of this work. Through the development of an innovative validation subroutine based on a permutation approach and the use of external information (i.e., operons), lack of a priori knowledge for undetected proteins was overcome. The integrative methodologies allowed for the identification of undetected proteins for Desulfovibrio vulgaris and Shewanella oneidensis for further biological exploration in laboratories towards finding functional relationships. In an effort to better understand diseases such as cancer at different developmental stages, the Microscale Life Science Center headquartered at the Arizona State University is pursuing single-cell studies by developing novel technologies. This research arranged and applied a statistical framework that tackled the following challenges: random noise, heterogeneous dynamic systems with multiple states, and understanding cell behavior within and across different Barrett's esophageal epithelial cell lines using oxygen consumption curves. These curves were characterized with good empirical fit using nonlinear models with simple structures which allowed extraction of a large number of features. Application of a supervised classification model to these features and the integration of experimental factors allowed for identification of subtle patterns among different cell types visualized through multidimensional scaling. Motivated by the challenges of analyzing real-time measurements, we further explored a unique two-dimensional representation of multiple time series using a wavelet approach which showcased promising results towards less complex approximations. Also, the benefits of external information were explored to improve the image representation.
ContributorsTorres Garcia, Wandaliz (Author) / Meldrum, Deirdre R. (Thesis advisor) / Runger, George C. (Thesis advisor) / Gel, Esma S. (Committee member) / Li, Jing (Committee member) / Zhang, Weiwen (Committee member) / Arizona State University (Publisher)
Created2011
Description
Immunosignaturing is a new immunodiagnostic technology that uses random-sequence peptide microarrays to profile the humoral immune response. Though the peptides have little sequence homology to any known protein, binding of serum antibodies may be detected, and the pattern correlated to disease states. The aim of my dissertation is to analyze the factors affecting the binding patterns using monoclonal antibodies and determine how much information may be extracted from the sequences. Specifically, I examined the effects of antibody concentration, competition, peptide density, and antibody valence. Peptide binding could be detected at the low concentrations relevant to immunosignaturing, and a monoclonal's signature could even be detected in the presences of 100 fold excess naive IgG. I also found that peptide density was important, but this effect was not due to bivalent binding. Next, I examined in more detail how a polyreactive antibody binds to the random sequence peptides compared to protein sequence derived peptides, and found that it bound to many peptides from both sets, but with low apparent affinity. An in depth look at how the peptide physicochemical properties and sequence complexity revealed that there were some correlations with properties, but they were generally small and varied greatly between antibodies. However, on a limited diversity but larger peptide library, I found that sequence complexity was important for antibody binding. The redundancy on that library did enable the identification of specific sub-sequences recognized by an antibody. The current immunosignaturing platform has little repetition of sub-sequences, so I evaluated several methods to infer antibody epitopes. I found two methods that had modest prediction accuracy, and I developed a software application called GuiTope to facilitate the epitope prediction analysis. None of the methods had sufficient accuracy to identify an unknown antigen from a database. In conclusion, the characteristics of the immunosignaturing platform observed through monoclonal antibody experiments demonstrate its promise as a new diagnostic technology. However, a major limitation is the difficulty in connecting the signature back to the original antigen, though larger peptide libraries could facilitate these predictions.
ContributorsHalperin, Rebecca (Author) / Johnston, Stephen A. (Thesis advisor) / Bordner, Andrew (Committee member) / Taylor, Thomas (Committee member) / Stafford, Phillip (Committee member) / Arizona State University (Publisher)
Created2011
Description
The oceans play an essential role in global biogeochemical cycles and in regulating climate. The biological carbon pump, the photosynthetic fixation of carbon dioxide by phytoplankton and subsequent sequestration of organic carbon into deep water, combined with the physical carbon pump, make the oceans the only long-term net sink for anthropogenic carbon dioxide. A full understanding of the workings of the biological carbon pump requires a knowledge of the role of different taxonomic groups of phytoplankton (protists and cyanobacteria) to organic carbon export. However, this has been difficult due to the degraded nature of particles sinking into particle traps, the main tools employed by oceanographers to collect sinking particulate matter in the ocean. In this study DNA-based molecular methods, including denaturing gradient gel electrophoresis, cloning and sequencing, and taxon-specific quantitative PCR, allowed for the first time for the identification of which protists and cyanobacteria contributed to the material collected by the traps in relation to their presence in the euphotic zone. I conducted this study at two time-series stations in the subtropical North Atlantic Ocean, one north of the Canary Islands, and one located south of Bermuda. The Bermuda study allowed me to investigate seasonal and interannual changes in the contribution of the plankton community to particle flux. I could also show that small unarmored taxa, including representatives of prasinophytes and cyanobacteria, constituted a significant fraction of sequences recovered from sediment trap material. Prasinophyte sequences alone could account for up to 13% of the clone library sequences of trap material during bloom periods. These observations contradict a long-standing paradigm in biological oceanography that only large taxa with mineral shells are capable of sinking while smaller, unarmored cells are recycled in the euphotic zone through the microbial loop. Climate change and a subsequent warming of the surface ocean may lead to a shift in the protist community toward smaller cell size in the future, but in light of these findings these changes may not necessarily lead to a reduction in the strength of the biological carbon pump.
ContributorsAmacher, Jessica (Author) / Neuer, Susanne (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Lomas, Michael (Committee member) / Wojciechowski, Martin (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2011
Description
African Swine Fever (ASF), endemic in many African countries, is now spreading to other continents. Though ASF is capable of incurring serious economic losses in affected countries, no vaccine exists to provide immunity to animals. Disease control relies largely on rapid diagnosis and the implementation of movement restrictions and strict eradication programs. Developing a scalable, accurate and low cost diagnostic for ASF will be of great help for the current situation. CIM's 10K random peptide microarray is a new high-throughput platform that allows systematic investigations of immune responses associated with disease and shows promise as a diagnostic tool. In this study, this new technology was applied to characterize the immune responses of ASF virus (ASFV) infections and immunizations. Six sets of sera from ASFV antigen immunized pigs, 6 sera from infected pigs and 20 sera samples from unexposed pigs were tested and analyzed statistically. Results show that both ASFV antigen immunized pigs and ASFV viral infected pigs can be distinguished from unexposed pigs. Since it appears that immune responses to other viral infections are also distinguishable on this platform, it holds the potential of being useful in developing a new ASF diagnostic. The ability of this platform to identify specific ASFV antibody epitopes was also explored. A subtle motif was found to be shared among a set of peptides displaying the highest reactivity for an antigen specific antibody. However, this motif does not seem to match with any antibody epitopes predicted by a linear antibody epitope prediction.
ContributorsXiao, Liang (Author) / Sykes, Kathryn (Thesis advisor) / Zhao, Zhan-Gong (Committee member) / Stafford, Phillip (Committee member) / Arizona State University (Publisher)
Created2011
Description
This thesis research focuses on developing a single-cell gene expression analysis method for marine diatom Thalassiosira pseudonana and constructing a chip level tool to realize the single cell RT-qPCR analysis. This chip will serve as a conceptual foundation for future deployable ocean monitoring systems. T. pseudonana, which is a common surface water microorganism, was detected in the deep ocean as confirmed by phylogenetic and microbial community functional studies. Six-fold copy number differences between 23S rRNA and 23S rDNA were observed by RT-qPCR, demonstrating the moderate functional activity of detected photosynthetic microbes in the deep ocean including T. pseudonana. Because of the ubiquity of T. pseudonana, it is a good candidate for an early warning system for ocean environmental perturbation monitoring. This early warning system will depend on identifying outlier gene expression at the single-cell level. An early warning system based on single-cell analysis is expected to detect environmental perturbations earlier than population level analysis which can only be observed after a whole community has reacted. Preliminary work using tube-based, two-step RT-qPCR revealed for the first time, gene expression heterogeneity of T. pseudonana under different nutrient conditions. Heterogeneity was revealed by different gene expression activity for individual cells under the same conditions. This single cell analysis showed a skewed, lognormal distribution and helped to find outlier cells. The results indicate that the geometric average becomes more important and representative of the whole population than the arithmetic average. This is in contrast with population level analysis which is limited to arithmetic averages only and highlights the value of single cell analysis. In order to develop a deployable sensor in the ocean, a chip level device was constructed. The chip contains surface-adhering droplets, defined by hydrophilic patterning, that serve as real-time PCR reaction chambers when they are immersed in oil. The chip had demonstrated sensitivities at the single cell level for both DNA and RNA. The successful rate of these chip-based reactions was around 85%. The sensitivity of the chip was equivalent to published microfluidic devices with complicated designs and protocols, but the production process of the chip was simple and the materials were all easily accessible in conventional environmental and/or biology laboratories. On-chip tests provided heterogeneity information about the whole population and were validated by comparing with conventional tube based methods and by p-values analysis. The power of chip-based single-cell analyses were mainly between 65-90% which were acceptable and can be further increased by higher throughput devices. With this chip and single-cell analysis approaches, a new paradigm for robust early warning systems of ocean environmental perturbation is possible.
ContributorsShi, Xu (Author) / Meldrum, Deirdre R. (Thesis advisor) / Zhang, Weiwen (Committee member) / Chao, Shih-hui (Committee member) / Westerhoff, Paul (Committee member) / Arizona State University (Publisher)
Created2013
Description
Microbial electrochemical cells (MXCs) are promising platforms for bioenergy production from renewable resources. In these systems, specialized anode-respiring bacteria (ARB) deliver electrons from oxidation of organic substrates to the anode of an MXC. While much progress has been made in understanding the microbiology, physiology, and electrochemistry of well-studied model ARB such as Geobacter and Shewanella, tremendous potential exists for MXCs as microbiological platforms for exploring novel ARB. This dissertation introduces approaches for selective enrichment and characterization of phototrophic, halophilic, and alkaliphilic ARB. An enrichment scheme based on manipulation of poised anode potential, light, and nutrient availability led to current generation that responded negatively to light. Analysis of phototrophically enriched communities suggested essential roles for green sulfur bacteria and halophilic ARB in electricity generation. Reconstruction of light-responsive current generation could be successfully achieved using cocultures of anode-respiring Geobacter and phototrophic Chlorobium isolated from the MXC enrichments. Experiments lacking exogenously supplied organic electron donors indicated that Geobacter could produce a measurable current from stored photosynthate in the dark. Community analysis of phototrophic enrichments also identified members of the novel genus Geoalkalibacter as potential ARB. Electrochemical characterization of two haloalkaliphilic, non-phototrophic Geoalkalibacter spp. showed that these bacteria were in fact capable of producing high current densities (4-8 A/m2) and using higher organic substrates under saline or alkaline conditions. The success of these selective enrichment approaches and community analyses in identifying and understanding novel ARB capabilities invites further use of MXCs as robust platforms for fundamental microbiological investigations.
ContributorsBadalamenti, Jonathan P (Author) / Krajmalnik-Brown, Rosa (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Rittmann, Bruce E. (Committee member) / Torres, César I (Committee member) / Vermaas, Willem (Committee member) / Arizona State University (Publisher)
Created2013