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- Genre: Academic theses
- Genre: Masters Thesis
- Creators: Borges, Chad
- Member of: ASU Electronic Theses and Dissertations
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Description
Efficient separation techniques for organelles and bacteria in the micron- and sub-micron range are required for various analytical challenges. Mitochondria have a wide size range resulting from the sub-populations, some of which may be associated with diseases or aging. However, traditional methods can often not resolve within-species size variations. Strategies to separate mitochondrial sub-populations by size are thus needed to study the importance of this organelle in cellular functions. Additionally, challenges also exist in distinguishing the sub-populations of bio-species which differ in the surface charge while possessing similar size, such as Salmonella typhimurium (Salmonella). The surface charge of Salmonella wild-type is altered upon environmental stimulations, influencing the bacterial survival and virulence within the host tissue. Therefore, it is important to explore methods to identify the sub-populations of Salmonella.
This work exploits insulator-based dielectrophoresis (iDEP) for the manipulation of mitochondria and Salmonella. The iDEP migration and trapping of mitochondria were investigated under both DC and low-frequency AC conditions, establishing that mitochondria exhibit negative DEP. Also, the first realization of size-based iDEP sorting experiments of mitochondria were demonstrated. As for Salmonella, the preliminary study revealed positive DEP behavior. Distinct trapping potential thresholds were found for the sub-populations with different surface charges.
Further, DEP was integrated with a non-intuitive migration mechanism termed absolute negative mobility (ANM), inducing a deterministic trapping component which allows the directed transport of µm- and sub-µm sized (bio)particles in microfluidic devices with a nonlinear post array under the periodic action of electrokinetic and dielectrophoretic forces. Regimes were revealed both numerically and experimentally in which larger particles migrate against the average applied force, whereas smaller particles show normal response. Moreover, this deterministic ANM (dANM) was characterized with polystyrene beads demonstrating improved migration speed at least two orders of magnitude higher compared to previous ANM systems with similar sized colloids. In addition, dANM was induced for mitochondria with an AC-overlaid waveform representing the first demonstration of ANM migration with biological species. Thus, it is envisioned that the efficient size selectivity of this novel migration mechanism can be employed in nanotechnology, organelle sub-population studies or fractionating protein nanocrystals.
This work exploits insulator-based dielectrophoresis (iDEP) for the manipulation of mitochondria and Salmonella. The iDEP migration and trapping of mitochondria were investigated under both DC and low-frequency AC conditions, establishing that mitochondria exhibit negative DEP. Also, the first realization of size-based iDEP sorting experiments of mitochondria were demonstrated. As for Salmonella, the preliminary study revealed positive DEP behavior. Distinct trapping potential thresholds were found for the sub-populations with different surface charges.
Further, DEP was integrated with a non-intuitive migration mechanism termed absolute negative mobility (ANM), inducing a deterministic trapping component which allows the directed transport of µm- and sub-µm sized (bio)particles in microfluidic devices with a nonlinear post array under the periodic action of electrokinetic and dielectrophoretic forces. Regimes were revealed both numerically and experimentally in which larger particles migrate against the average applied force, whereas smaller particles show normal response. Moreover, this deterministic ANM (dANM) was characterized with polystyrene beads demonstrating improved migration speed at least two orders of magnitude higher compared to previous ANM systems with similar sized colloids. In addition, dANM was induced for mitochondria with an AC-overlaid waveform representing the first demonstration of ANM migration with biological species. Thus, it is envisioned that the efficient size selectivity of this novel migration mechanism can be employed in nanotechnology, organelle sub-population studies or fractionating protein nanocrystals.
ContributorsLuo, Jinghui (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2015
Description
Cancer is a heterogeneous disease with discrete oncogenic mechanisms. P53 mutation is the most common oncogenic mutation in many cancers including breast cancer. This dissertation focuses on fundamental genetic alterations enforced by p53 mutation as an indirect target. p53 mutation upregulates the mevalonate pathway genes altering cholesterol biosynthesis and prenylation. Prenylation, a lipid modification, is required for small GTPases signaling cascades. Project 1 demonstrates that prenylation inhibition can specifically target cells harboring p53 mutation resulting in reduced tumor proliferation and migration. Mutating p53 is associated with Ras and RhoA activation and statin prevents this activity by inhibiting prenylation. Ras-related pathway genes were selected from the transcriptomic analysis for evaluating correlation to statin sensitivity. A gene signature of seventeen genes and TP53 genotype (referred to as MPR signature) is generated to predict response to statins. MPR signature is validated through two datasets of drug screening in cell lines. As advancements in targeted gene modification are rising, the CRISPR-Cas9 technology has emerged as a new cancer therapeutic strategy. One of the important risk factors in gene therapy is the immune recognition of the exogenous therapeutic tool, resulting in obstruction of treatment and possibly serious health consequences. Project 2 describes a method development that can potentially improve the safety and efficacy of gene-targeting proteins. A cohort of 155 healthy individuals was screened for pre-existing B cell and T cell immune response to the S. pyogenes Cas9 protein. We detected antibodies against Cas9 in more than 10% of the healthy population and identified two immunodominant T cell epitopes of this protein. A de-immunized Cas9 that maintains the wild-type functionality was engineered by mutating the identified T cell epitopes. The gene signature and method described here have the potential to improve strategies for genome-driven tumor targeting.
ContributorsRoshdi Ferdosi, Shayesteh (Author) / Anderson, Karen S (Thesis advisor) / LaBaer, Joshua (Thesis advisor) / Woodbury, Neel (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2017
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
DNA, RNA and Protein are three pivotal biomolecules in human and other organisms, playing decisive roles in functionality, appearance, diseases development and other physiological phenomena. Hence, sequencing of these biomolecules acquires the prime interest in the scientific community. Single molecular identification of their building blocks can be done by a technique called Recognition Tunneling (RT) based on Scanning Tunneling Microscope (STM). A single layer of specially designed recognition molecule is attached to the STM electrodes, which trap the targeted molecules (DNA nucleoside monophosphates, RNA nucleoside monophosphates or amino acids) inside the STM nanogap. Depending on their different binding interactions with the recognition molecules, the analyte molecules generate stochastic signal trains accommodating their “electronic fingerprints”. Signal features are used to detect the molecules using a machine learning algorithm and different molecules can be identified with significantly high accuracy. This, in turn, paves the way for rapid, economical nanopore sequencing platform, overcoming the drawbacks of Next Generation Sequencing (NGS) techniques.
To read DNA nucleotides with high accuracy in an STM tunnel junction a series of nitrogen-based heterocycles were designed and examined to check their capabilities to interact with naturally occurring DNA nucleotides by hydrogen bonding in the tunnel junction. These recognition molecules are Benzimidazole, Imidazole, Triazole and Pyrrole. Benzimidazole proved to be best among them showing DNA nucleotide classification accuracy close to 99%. Also, Imidazole reader can read an abasic monophosphate (AP), a product from depurination or depyrimidination that occurs 10,000 times per human cell per day.
In another study, I have investigated a new universal reader, 1-(2-mercaptoethyl)pyrene (Pyrene reader) based on stacking interactions, which should be more specific to the canonical DNA nucleosides. In addition, Pyrene reader showed higher DNA base-calling accuracy compare to Imidazole reader, the workhorse in our previous projects. In my other projects, various amino acids and RNA nucleoside monophosphates were also classified with significantly high accuracy using RT. Twenty naturally occurring amino acids and various RNA nucleosides (four canonical and two modified) were successfully identified. Thus, we envision nanopore sequencing biomolecules using Recognition Tunneling (RT) that should provide comprehensive betterment over current technologies in terms of time, chemical and instrumental cost and capability of de novo sequencing.
To read DNA nucleotides with high accuracy in an STM tunnel junction a series of nitrogen-based heterocycles were designed and examined to check their capabilities to interact with naturally occurring DNA nucleotides by hydrogen bonding in the tunnel junction. These recognition molecules are Benzimidazole, Imidazole, Triazole and Pyrrole. Benzimidazole proved to be best among them showing DNA nucleotide classification accuracy close to 99%. Also, Imidazole reader can read an abasic monophosphate (AP), a product from depurination or depyrimidination that occurs 10,000 times per human cell per day.
In another study, I have investigated a new universal reader, 1-(2-mercaptoethyl)pyrene (Pyrene reader) based on stacking interactions, which should be more specific to the canonical DNA nucleosides. In addition, Pyrene reader showed higher DNA base-calling accuracy compare to Imidazole reader, the workhorse in our previous projects. In my other projects, various amino acids and RNA nucleoside monophosphates were also classified with significantly high accuracy using RT. Twenty naturally occurring amino acids and various RNA nucleosides (four canonical and two modified) were successfully identified. Thus, we envision nanopore sequencing biomolecules using Recognition Tunneling (RT) that should provide comprehensive betterment over current technologies in terms of time, chemical and instrumental cost and capability of de novo sequencing.
ContributorsSen, Suman (Author) / Lindsay, Stuart (Thesis advisor) / Zhang, Peiming (Thesis advisor) / Gould, Ian R. (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2016
Description
For reading DNA bases more accurately, a series of nitrogen-containing aromatic heterocycles have been designed and synthesized as candidates of universal reader to interact with all naturally occurring DNA nucleobases by hydrogen bonding interaction and eventually is used to read DNA by recognition tunneling. These recognition molecules include 6-mercapto-1H-benzo[d]imidazole-2-carboxamide, 5-(2-mercaptoethyl)-1H-imidazole-2-carboxamide, 5-(2-mercaptoethyl)-4H-1,2,4-traizole-3-carboxamide and 1-(2-mercaptoethyl)-1H-pyrrole-3-carboxamide. Their formation of hydrogen bonding complexes with nucleobases was studied and association constants were measured by proton NMR titration experiments in deuterated chloroform at room temperature. To do so, the mercaptoethyl chain or thiol group of these reading molecules was replaced or protected with the more lipophilic group to increase the solubility of these candidates in CDCl3. The 3' and 5' hydroxyl groups of deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC) and thymidine (dT) were protected with tert-butyldimethylsilyl (TBDMS) to eliminate hydrogen bonding competition from the hydroxyl protons with these candidates as well as to increase the solubility of the nucleosides in CDCl3 for NMR titration experiment. Benzimidazole and imidazole containing readers exhibited the strongest H-bonding affinity towards DNA bases where pyrrole containing reader showed the weakest affinity. In all cases, dG revealed the strongest affinity towards the readers while dA showed the least.
The molecular complex formation in aqueous solution was studied by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry. The formation of both 1:1 and 2:1 complexes between one or two reading molecules and a DNA nucleotide were observed by ESI mass. A series of amino acids and carbohydrates were also examined by mass spectrometry to show the formation of non-covalent complexes with imidazole reader in aqueous solution. The experimental results were compared by calculating energies of ground state conformers of individual molecules and their complexes using computer modeling study by DFT calculations. These studies give insights into the molecular interactions that happen in a nanogap during recognition tunneling experiments.
The molecular complex formation in aqueous solution was studied by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry. The formation of both 1:1 and 2:1 complexes between one or two reading molecules and a DNA nucleotide were observed by ESI mass. A series of amino acids and carbohydrates were also examined by mass spectrometry to show the formation of non-covalent complexes with imidazole reader in aqueous solution. The experimental results were compared by calculating energies of ground state conformers of individual molecules and their complexes using computer modeling study by DFT calculations. These studies give insights into the molecular interactions that happen in a nanogap during recognition tunneling experiments.
ContributorsBiswas, Sovan (Author) / Lindsay, Stuart (Thesis advisor) / Zhang, Peiming (Thesis advisor) / Borges, Chad (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2016
Description
In species with highly heteromorphic sex chromosomes, the degradation of one of the sex chromosomes can result in unequal gene expression between the sexes (e.g., between XX females and XY males) and between the sex chromosomes and the autosomes. Dosage compensation is a process whereby genes on the sex chromosomes achieve equal gene expression which prevents deleterious side effects from having too much or too little expression of genes on sex chromsomes. The green anole is part of a group of species that recently underwent an adaptive radiation. The green anole has XX/XY sex determination, but the content of the X chromosome and its evolution have not been described. Given its status as a model species, better understanding the green anole genome could reveal insights into other species. Genomic analyses are crucial for a comprehensive picture of sex chromosome differentiation and dosage compensation, in addition to understanding speciation.
In order to address this, multiple comparative genomics and bioinformatics analyses were conducted to elucidate patterns of evolution in the green anole and across multiple anole species. Comparative genomics analyses were used to infer additional X-linked loci in the green anole, RNAseq data from male and female samples were anayzed to quantify patterns of sex-biased gene expression across the genome, and the extent of dosage compensation on the anole X chromosome was characterized, providing evidence that the sex chromosomes in the green anole are dosage compensated.
In addition, X-linked genes have a lower ratio of nonsynonymous to synonymous substitution rates than the autosomes when compared to other Anolis species, and pairwise rates of evolution in genes across the anole genome were analyzed. To conduct this analysis a new pipeline was created for filtering alignments and performing batch calculations for whole genome coding sequences. This pipeline has been made publicly available.
In order to address this, multiple comparative genomics and bioinformatics analyses were conducted to elucidate patterns of evolution in the green anole and across multiple anole species. Comparative genomics analyses were used to infer additional X-linked loci in the green anole, RNAseq data from male and female samples were anayzed to quantify patterns of sex-biased gene expression across the genome, and the extent of dosage compensation on the anole X chromosome was characterized, providing evidence that the sex chromosomes in the green anole are dosage compensated.
In addition, X-linked genes have a lower ratio of nonsynonymous to synonymous substitution rates than the autosomes when compared to other Anolis species, and pairwise rates of evolution in genes across the anole genome were analyzed. To conduct this analysis a new pipeline was created for filtering alignments and performing batch calculations for whole genome coding sequences. This pipeline has been made publicly available.
ContributorsRupp, Shawn Michael (Author) / Wilson Sayres, Melissa A (Thesis advisor) / Kusumi, Kenro (Committee member) / DeNardo, Dale (Committee member) / Arizona State University (Publisher)
Created2016
Description
In this thesis, a breadboard Integrated Microarray Printing and Detection System (IMPDS) was proposed to address key limitations of traditional microarrays. IMPDS integrated two core components of a high-resolution surface plasmon resonance imaging (SPRi) system and a piezoelectric dispensing system that can print ultra-low volume droplets. To avoid evaporation of droplets in the microarray, a 100 μm thick oil layer (dodecane) was used to cover the chip surface. The interaction between BSA (Bovine serum albumin) and Anti-BSA was used to evaluate the capability of IMPDS. The alignment variability of printing, stability of droplets array and quantification of protein-protein interactions based on nanodroplet array were evaluated through a 10 x 10 microarray on SPR sensor chip. Binding kinetic constants obtained from IMPDS are close with results from commercial SPR setup (BI-3000), which indicates that IMPDS is capable to measure kinetic constants accurately. The IMPDS setup has following advantages: 1) nanoliter scale sample consumption, 2) high-throughput detection with real-time kinetic information for biomolecular interactions, 3) real-time information during printing and spot-on-spot detection of biomolecular interactions 4) flexible selection of probes and receptors (M x N interactions). Since IMPDS studies biomolecular interactions with low cost and high flexibility in real-time manner, it has great potential in applications such as drug discovery, food safety and disease diagnostics, etc.
ContributorsXiao, Feng (Author) / Tao, Nongjian (Thesis advisor) / Borges, Chad (Committee member) / Guo, Jia (Committee member) / Arizona State University (Publisher)
Created2017
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