Matching Items (8)
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- All Subjects: Biochemistry
- Genre: Doctoral Dissertation
- Creators: Borges, Chad
- Resource Type: Text
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
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
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
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
Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that represents the ancient fusion of two major thiol-disulfide oxidoreductase gene families: thioredoxin and ERV. QSOX1 was first linked with cancer after being identified as overexpressed in pancreatic ductal adenocarcinoma (but not in adjacent normal ductal epithelia, infiltrating lymphocytes, or chronic pancreatitis). QSOX1 overexpression has been confirmed in a number of other histological tumor types, such as breast, lung, kidney, prostate, and others. Expression of QSOX1 supports a proliferative and invasive phenotype in tumor cells, and its enzymatic activity is critical for promoting an invasive phenotype. An in vivo tumor growth study utilizing the pancreatic tumor cell line MIAPaCa-2 containing a QSOX1-silencing shRNA construct revealed that QSOX1 expression supports a proliferative phenotype. These preliminary studies suggest that suppressing the enzymatic activity of QSOX1 could represent a novel therapeutic strategy to inhibit proliferation and invasion of malignant neoplasms.
The goal of this research was to identify and characterize biologically active small molecule inhibitors for QSOX1. Chemical inhibition of QSOX1 enzymatic activity was hypothesized to reduce growth and invasion of tumor cells. Recombinant QSOX1 was screened against libraries of small molecules using an enzymatic activity assay to identify potential QSOX1 inhibitors. Two lead QSOX1 inhibitors were confirmed, 2-phenyl-1, 2-benzisoselenazol-3-one (ebselen), and 3-methoxy-n-[4-(1 pyrrolidinyl)phenyl]benzamide. The biological activity of these compounds is consistent with QSOX1 knockdown in tumor cell lines, reducing growth and invasion in vitro. Treatment of tumor cells with these compounds also resulted in specific ECM defects, a phenotype associated with QSOX1 knockdown. Additionally, these compounds were shown to be active in pancreatic and renal cancer xenografts, reducing tumor growth with daily treatment. For ebselen, the molecular mechanism of inhibition was determined using a combination of biochemical and mass spectrometric techniques. The results obtained in these studies provide proof-of-principle that targeting QSOX1 enzymatic activity with chemical compounds represents a novel potential therapeutic avenue worthy of further investigation in cancer. Additionally, the utility of these small molecules as chemical probes will yield future insight into the general biology of QSOX1, including the identification of novel substrates of QSOX1.
The goal of this research was to identify and characterize biologically active small molecule inhibitors for QSOX1. Chemical inhibition of QSOX1 enzymatic activity was hypothesized to reduce growth and invasion of tumor cells. Recombinant QSOX1 was screened against libraries of small molecules using an enzymatic activity assay to identify potential QSOX1 inhibitors. Two lead QSOX1 inhibitors were confirmed, 2-phenyl-1, 2-benzisoselenazol-3-one (ebselen), and 3-methoxy-n-[4-(1 pyrrolidinyl)phenyl]benzamide. The biological activity of these compounds is consistent with QSOX1 knockdown in tumor cell lines, reducing growth and invasion in vitro. Treatment of tumor cells with these compounds also resulted in specific ECM defects, a phenotype associated with QSOX1 knockdown. Additionally, these compounds were shown to be active in pancreatic and renal cancer xenografts, reducing tumor growth with daily treatment. For ebselen, the molecular mechanism of inhibition was determined using a combination of biochemical and mass spectrometric techniques. The results obtained in these studies provide proof-of-principle that targeting QSOX1 enzymatic activity with chemical compounds represents a novel potential therapeutic avenue worthy of further investigation in cancer. Additionally, the utility of these small molecules as chemical probes will yield future insight into the general biology of QSOX1, including the identification of novel substrates of QSOX1.
ContributorsHanavan, Paul D (Author) / Lake, Douglas (Thesis advisor) / LaBaer, Joshua (Committee member) / Mangone, Marco (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2015
Description
Spatial resolved detection and quantification of ribonucleic acid (RNA) molecules in single cell is crucial for the understanding of inherent biological issues, like mechanism of gene regulation or the development and maintenance of cell fate. Conventional methods for single cell RNA profiling, like single-cell RNA sequencing (scRNA-seq) or single-molecule fluorescent in situ hybridization (smFISH), suffer either from the loss of spatial information or the low detection throughput. In order to advance single-cell analysis, new approaches need to be developed with the ability to perform high-throughput detection while preserving spatial information of the subcellular location of target RNA molecules.
Novel approaches for highly multiplexed single cell in situ transcriptomic analysis were developed by our group to enable single-cell comprehensive RNA profiling in their native spatial contexts. Reiterative FISH was demonstrated to be able to detect >100 RNA species in single cell in situ, while more sophisticated approaches, consecutive FISH (C-FISH) and switchable fluorescent oligonucleotide based FISH (SFO-FISH), have the potential for whole transcriptome profiling at the single molecule sensitivity. The introduction of a cleavable fluorescent tyramide even enables sensitive RNA profiling in intact tissues with high throughput. These approaches will have wide applications in studies of systems biology, molecular diagnosis and targeted therapies.
Novel approaches for highly multiplexed single cell in situ transcriptomic analysis were developed by our group to enable single-cell comprehensive RNA profiling in their native spatial contexts. Reiterative FISH was demonstrated to be able to detect >100 RNA species in single cell in situ, while more sophisticated approaches, consecutive FISH (C-FISH) and switchable fluorescent oligonucleotide based FISH (SFO-FISH), have the potential for whole transcriptome profiling at the single molecule sensitivity. The introduction of a cleavable fluorescent tyramide even enables sensitive RNA profiling in intact tissues with high throughput. These approaches will have wide applications in studies of systems biology, molecular diagnosis and targeted therapies.
ContributorsXiao, Lu, Ph.D (Author) / Guo, Jia (Thesis advisor) / Wang, Xu (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2019
Description
Since the conception of DNA nanotechnology, the field has evolved towards the development of complex, dynamic 3D structures. The predictability of Watson-Crick base pairing makes DNA an unparalleled building block, and enables exceptional programmability in nanostructure shape and size. The work presented in this dissertation focuses on expanding two facets of the field: (1) introducing functionality through the incorporation of peptides to create DNA-peptide hybrid materials, and (2) the development of self-assembling DNA crystal lattices for scaffolding biomolecules. DNA nanostructures have long been proposed as drug delivery vehicles; however, they are not biocompatible because of their low stability in low salt environments and entrapment within the endosome. To address these issues, a functionalized peptide coating was designed to act as a counterion to a six-helix bundle, while simultaneously displaying numerous copies of an endosomal escape peptide to enable cytosolic delivery. This functionalized peptide coating creates a DNA-peptide hybrid material, but does not allow specific positioning or orientation of the peptides. The ability to control those aspects required the synthesis of DNA-peptide or DNA-peptide-DNA conjugates that can be incorporated into the nanostructure. The approach was utilized to produce a synbody where three peptides that bind transferrin with micromolar affinity, which were presented for multivalent binding to optimize affinity. Additionally, two DNA handle was attached to an enzymatically cleavable peptide to link two unique nanostructures. The second DNA handle was also used to constrain the peptide in a cyclic fashion to mimic the cell-adhesive conformations of RGD and PHSRN in fibronectin.
The original goal of DNA nanotechnology was to use a crystalline lattice made of DNA to host proteins for their structural determination using X-ray crystallography. The work presented here takes significant steps towards achieving this goal, including elucidating design rules to control cavity size within the scaffold for accommodating guest molecules of unique sizes, approaches to improve the atomic detail of the scaffold, and strategies to modulate the symmetry of each unique lattice. Finally, this work surveys methodologies towards the incorporation of several guest molecules, with promising preliminary results that constitute a significant advancement towards the ultimate goal of the field.
ContributorsMacCulloch, Tara Lynn (Author) / Stephanopoulos, Nicholas (Thesis advisor) / Borges, Chad (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2021
Description
Exerting bias on a diverse pool of random short single stranded oligonucleotides (ODNs) by favoring binding to a specific target has led to the identification of countless high affinity aptamers with specificity to a single target. By exerting this same bias without prior knowledge of targets generates libraries to capture the complex network of molecular interactions presented in various biological states such as disease or cancer. Aptamers and enriched libraries have vast applications in bio-sensing, therapeutics, targeted drug delivery, biomarker discovery, and assay development. Here I describe a novel method of computational biophysical characterization of molecular interactions between a single aptamer and its cognate target as well as an alternative to next generation sequencing (NGS) as a readout for a SELEX-based assay. I demonstrate the capability of an artificial neural network (ANN) trained on the results of screening an aptamer against a random sampling of a combinatorial library of short synthetic 11mer peptides to accurately predict the binding intensities of that aptamer to the remainder of the combinatorial space originally sampled. This machine learned comprehensive non-linear relationship between amino acid sequence and aptamer binding to synthetic peptides can also make biologically relevant predictions for probable molecular interactions between the aptamer and its cognate target. Results of SELEX-based assays are determined by quantifying the presence and frequency of informative species after probing patient specimen. Here I show the potential of DNA microarrays to simultaneously monitor a pool of informative sequences within a diverse library with similar variability and reproducibility as NGS.
ContributorsLevenberg, Symon (Author) / Woodbury, Neal (Thesis advisor) / Borges, Chad (Committee member) / Ghirlanda, Giovanna (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2021