Matching Items (70)
Description
Emerging pathogens present several challenges to medical diagnostics. Primarily, the exponential spread of a novel pathogen through naïve populations require a rapid and overwhelming diagnostic response at the site of outbreak. While point-of-care (PoC) platforms have been developed for detection of antigens, serologic responses, and pathogenic genomes, only nucleic acid diagnostics currently have the potential to be developed and manufactured within weeks of an outbreak owing to the speed of next-generation sequencing and custom DNA synthesis. Among nucleic acid diagnostics, isothermal amplification strategies are uniquely suited for PoC implementation due to their simple instrumentation and lack of thermocycling requirement. Unfortunately, isothermal strategies are currently prone to spurious nonspecific amplification, hindering their specificity and necessitating extensive empirical design pipelines that are both time and resource intensive. In this work, isothermal amplification strategies are extensively compared for their feasibility of implementation in outbreak response scenarios. One such technology, Loop-mediated Amplification (LAMP), is identified as having high-potential for rapid development and PoC deployment. Various approaches to abrogating nonspecific amplification are described including a novel in silico design tool based on coarse-grained simulation of interactions between thermophilic DNA polymerase and DNA strands in isothermal reaction conditions. Nonspecific amplification is shown to be due to stabilization of primer secondary structures by high concentrations of Bst DNA polymerase and a mechanism of micro-complement-mediated cross-priming is demonstrated as causal via nanopore sequencing of nonspecific reaction products. The resulting computational model predicts primer set background in 64% of 67 test assays and its usefulness is illustrated further by determining problematic primers in a West Nile Virus-specific LAMP primer set and optimizing primer 3’ nucleotides to eliminate micro-complements within the reaction, resulting in inhibition of background accumulation. Finally, the emergence of Orthopox monkeypox (MPXV) as a recurring threat is discussed and SimCycle is utilized to develop a novel technique for clade-specific discrimination of MPXV based on bridging viral genomic rearrangements (Bridging LAMP). Bridging LAMP is implemented in a 4-plex microfluidic format and demonstrates 100% sensitivity in detection of 100 copies of viral lysates and 45 crude MPXV-positive patient samples collected during the 2022 Clade IIb outbreak.
ContributorsKnappenberger, Mark Daniel (Author) / Anderson, Karen S (Thesis advisor) / LaBaer, Joshua (Committee member) / Roberson, Robert (Committee member) / Lindsay, Stuart (Committee member) / Arizona State University (Publisher)
Created2023
Description
MicroRNAs (miRNAs) are 17-22 nucleotide non-coding RNAs that regulate gene expression by targeting non-complementary elements in the 3’ untranslated regions (3’UTRs) of mRNAs. miRNAs, which form complex networks of interaction that differ by tissue and developmental stage, display conservation in their function across metazoan species. Yet much remains unknown regarding their biogenesis, localization, strand selection, and their absolute abundance due to the difficulty of detecting and amplifying such small molecules. Here, I used an updated HT qPCR-based methodology to follow miRNA expression of 5p and 3p strands for all 190 C. elegans miRNAs described in miRBase throughout all six developmental stages in triplicates (total of 9,708 experiments), and studied their expression levels, tissue localization, and the rules underlying miRNA strand selection. My study validated previous findings and identified novel, conserved patterns of miRNA strand expression throughout C. elegans development, which at times correlate with previously observed developmental phenotypes. Additionally, my results highlighted novel structural principles underlying strand selection, which can be applied to higher metazoans.
Though optimized for use in C. elegans, this method can be easily adapted to other eukaryotic systems, allowing for more scalable quantitative investigation of miRNA biology and/or miRNA diagnostics.
ContributorsMeadows, Dalton Alexander (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Joshua (Committee member) / Murugan, Vel (Committee member) / Wilson-Rawls, Jeanne (Committee member) / Arizona State University (Publisher)
Created2023
Description
Under the direction of Dr. Carolyn Compton, a group of seven Barrett honors students have embarked on a truly unique team thesis project to create a documentary on the process of creating a COVID-19 testing laboratory. This documentary tells the story of the ASU Biodesign Clinical Testing Laboratory (ABCTL), the first lab in the western United States to offer public saliva testing to identify the presence of COVID-19.
ContributorsCura, Joriel (Director, Photographer) / Foote, Hannah (Producer, Sound designer) / Raymond, Julia (Production personnel) / Bardfeld, Sierra (Narrator, Editor) / Dholaria, Nikhil (Writer of added commentary) / Liu, Tara (Writer of added commentary) / Varghese, Mahima (Writer of added commentary) / Compton, Carolyn C. (Interviewee, Project director) / Harris, Valerie (Interviewee) / LaBaer, Joshua (Interviewee) / Miceli, Joseph (Interviewee) / Nelson, Megan (Interviewee) / Ungaro, Brianna (Interviewee)
Created2021
Description
Signal transduction networks comprising protein-protein interactions (PPIs) mediate homeostatic, diseased, and therapeutic cellular responses. Mapping these networks has primarily focused on identifying interactors, but less is known about the interaction affinity, rates of interaction or their regulation. To better understand the extent of the annotated human interactome, I first examined > 2500 protein interactions within the B cell receptor (BCR) signaling pathway using a current, cutting-edge bioluminescence-based platform called “NanoBRET” that is capable of analyzing transient and stable interactions in high throughput. Eighty-three percent (83%) of the detected interactions have not been previously reported, indicating that much of the BCR pathway is still unexplored. Unfortunately, NanoBRET, as with all other high throughput methods, cannot determine binding kinetics or affinities. To address this shortcoming, I developed a hybrid platform that characterizes > 400 PPIs quantitatively and simultaneously in < 1 hour by combining the high throughput and flexible nature of nucleic programmable protein arrays (NAPPA) with the quantitative abilities of surface plasmon resonance imaging (SPRi). NAPPA-SPRi was then used to study the kinetics and affinities of > 12,000 PPIs in the BCR signaling pathway, revealing unique kinetic mechanisms that are employed by proteins, phosphorylation and activation states to regulate PPIs. In one example, activation of the GTPase RAC1 with nonhydrolyzable GTP-γS minimally affected its binding affinities with phosphorylated proteins but increased, on average, its on- and off-rates by 4 orders of magnitude for one-third of its interactions. In contrast, this phenomenon occurred with virtually all unphosphorylated proteins. The majority of the interactions (85%) were novel, sharing 40% of the same interactions as NanoBRET as well as detecting 55% more interactions than NanoBRET. In addition, I further validated four novel interactions identified by NAPPA-SPRi using SDS-PAGE migration and Western blot analyses. In one case, we have the first evidence of a direct enzyme-substrate interaction between two well-known proto-oncogenes that are abnormally regulated in > 30% of cancers, PI3K and MYC. Herein, PI3K is demonstrated to phosphorylate MYC at serine 62, a phosphosite that increases the stability of MYC. This study provides valuable insight into how PPIs, phosphorylation, and GTPase activation regulate the BCR signal transduction pathway. In addition, these methods could be applied toward understanding other signaling pathways, pathogen-host interactions, and the effect of protein mutations on protein interactions.
ContributorsPetritis, Brianne Ogata (Author) / LaBaer, Joshua (Thesis advisor) / Lake, Douglas (Committee member) / Wang, Shaopeng (Committee member) / Arizona State University (Publisher)
Created2018
Description
According to the World Health Organization, cancer is one of the leading causes of death around the world. Although early diagnostics using biomarkers and improved treatments with targeted therapy have reduced the rate of cancer related mortalities, there remain many unknowns regarding the contributions of the tumor microenvironment to cancer progression and therapeutic resistance. The tumor microenvironment plays a significant role by manipulating the progression of cancer cells through biochemical and biophysical signals from the surrounding stromal cells along with the extracellular matrix. As such, there is a critical need to understand how the tumor microenvironment influences the molecular mechanisms underlying cancer metastasis to facilitate the discovery of better therapies. This thesis described the development of microfluidic technologies to study the interplay of cancer cells with their surrounding microenvironment. The microfluidic model was used to assess how exposure to chemoattractant, epidermal growth factor (EGF), impacted 3D breast cancer cell invasion and enhanced cell motility speed was noted in the presence of EGF validating physiological cell behavior. Additionally, breast cancer and patient-derived cancer-associated fibroblast (CAF) cells were co-cultured to study cell-cell crosstalk and how it affected cancer invasion. GPNMB was identified as a novel gene of interest and it was shown that CAFs enhanced breast cancer invasion by up-regulating the expression of GPNMB on breast cancer cells resulting in increased migration speed. Lastly, this thesis described the design, biological validation, and use of this microfluidic platform as a new in vitro 3D organotypic model to study mechanisms of glioma stem cell (GSC) invasion in the context of a vascular niche. It was confirmed that CXCL12-CXCR4 signaling is involved in promoting GSC invasion in a 3D vascular microenvironment, while also demonstrating the effectiveness of the microfluidic as a drug screening assay. Taken together, the broader impacts of the microfluidic model developed in this dissertation include, a possible alternative platform to animal testing that is focused on mimicking human physiology, a potential ex vivo platform using patient-derived cells for studying the interplay of cancer cells with its surrounding microenvironment, and development of future therapeutic strategies tailored toward disrupting key molecular pathways involved in regulatory mechanisms of cancer invasion.
ContributorsTruong, Danh, Ph.D (Author) / Nikkhah, Mehdi (Thesis advisor) / LaBaer, Joshua (Committee member) / Smith, Barbara (Committee member) / Mouneimne, Ghassan (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2018
Description
Multicellular organisms use precise gene regulation, executed throughout development, to build and sustain various cell and tissue types. Post-transcriptional gene regulation is essential for metazoan development and acts on mRNA to determine its localization, stability, and translation. MicroRNAs (miRNAs) and RNA binding proteins (RBPs) are the principal effectors of post-transcriptional gene regulation and act by targeting the 3'untranslated regions (3'UTRs) of mRNA. MiRNAs are small non-coding RNAs that have the potential to regulate hundreds to thousands of genes and are dysregulated in many prevalent human diseases such as diabetes, Alzheimer's disease, Duchenne muscular dystrophy, and cancer. However, the precise contribution of miRNAs to the pathology of these diseases is not known.
MiRNA-based gene regulation occurs in a tissue-specific manner and is implemented by an interplay of poorly understood and complex mechanisms, which control both the presence of the miRNAs and their targets. As a consequence, the precise contributions of miRNAs to gene regulation are not well known. The research presented in this thesis systematically explores the targets and effects of miRNA-based gene regulation in cell lines and tissues.
I hypothesize that miRNAs have distinct tissue-specific roles that contribute to the gene expression differences seen across tissues. To address this hypothesis and expand our understanding of miRNA-based gene regulation, 1) I developed the human 3'UTRome v1, a resource for studying post-transcriptional gene regulation. Using this resource, I explored the targets of two cancer-associated miRNAs miR-221 and let-7c. I identified novel targets of both these miRNAs, which present potential mechanisms by which they contribute to cancer. 2) Identified in vivo, tissue-specific targets in the intestine and body muscle of the model organism Caenorhabditis elegans. The results from this study revealed that miRNAs regulate tissue homeostasis, and that alternative polyadenylation and miRNA expression patterns modulate miRNA targeting at the tissue-specific level. 3) Explored the functional relevance of miRNA targeting to tissue-specific gene expression, where I found that miRNAs contribute to the biogenesis of mRNAs, through alternative splicing, by regulating tissue-specific expression of splicing factors. These results expand our understanding of the mechanisms that guide miRNA targeting and its effects on tissue-specific gene expression.
MiRNA-based gene regulation occurs in a tissue-specific manner and is implemented by an interplay of poorly understood and complex mechanisms, which control both the presence of the miRNAs and their targets. As a consequence, the precise contributions of miRNAs to gene regulation are not well known. The research presented in this thesis systematically explores the targets and effects of miRNA-based gene regulation in cell lines and tissues.
I hypothesize that miRNAs have distinct tissue-specific roles that contribute to the gene expression differences seen across tissues. To address this hypothesis and expand our understanding of miRNA-based gene regulation, 1) I developed the human 3'UTRome v1, a resource for studying post-transcriptional gene regulation. Using this resource, I explored the targets of two cancer-associated miRNAs miR-221 and let-7c. I identified novel targets of both these miRNAs, which present potential mechanisms by which they contribute to cancer. 2) Identified in vivo, tissue-specific targets in the intestine and body muscle of the model organism Caenorhabditis elegans. The results from this study revealed that miRNAs regulate tissue homeostasis, and that alternative polyadenylation and miRNA expression patterns modulate miRNA targeting at the tissue-specific level. 3) Explored the functional relevance of miRNA targeting to tissue-specific gene expression, where I found that miRNAs contribute to the biogenesis of mRNAs, through alternative splicing, by regulating tissue-specific expression of splicing factors. These results expand our understanding of the mechanisms that guide miRNA targeting and its effects on tissue-specific gene expression.
ContributorsKotagama, Kasuen Indrajith Bandara (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Joshua (Committee member) / Newbern, Jason (Committee member) / Rawls, Alan (Committee member) / Arizona State University (Publisher)
Created2019
Description
Biomarkers find a wide variety of applications in oncology from risk assessment to diagnosis and predicting and monitoring recurrence and response to therapy. Developing clinically useful biomarkers for cancer is faced with several challenges, including cancer heterogeneity and factors related to assay development and biomarker performance. Circulating biomarkers offer a rapid, cost-effective, and minimally-invasive window to disease and are ideal for population-based screening. Circulating immune biomarkers are stable, measurable, and can betray the underlying antigen when present below detection levels or even no longer present. This dissertation aims to investigate potential circulating immune biomarkers with applications in cancer detection and novel therapies. Over 600,000 cancers each year are attributed to the human papillomavirus (HPV), including cervical, anogenital and oropharyngeal cancers. A key challenge in understanding HPV immunobiology and developing immune biomarkers is the diversity of HPV types and the need for multiplexed display of HPV antigens. In Project 1, nucleic acid programmable protein arrays displaying the proteomes of 12 HPV types were developed and used for serum immunoprofiling of women with cervical lesions or invasive cervical cancer. These arrays provide a valuable high-throughput tool for measuring the breadth, specificity, heterogeneity, and cross-reactivity of the serologic response to HPV. Project 2 investigates potential biomarkers of immunity to the bacterial CRISPR/Cas9 system that is currently in clinical trials for cancer. Pre-existing B cell and T cell immune responses to Cas9 were detected in humans and Cas9 was modified to eliminate immunodominant epitopes while preserving its function and specificity. This dissertation broadens our understanding of the immunobiology of cervical cancer and provides insights into the immune profiles that could serve as biomarkers of various applications in cancer.
ContributorsEwaisha, Radwa Mohamed Emadeldin Mahmoud (Author) / Anderson, Karen S (Thesis advisor) / LaBaer, Joshua (Committee member) / Lake, Douglas F (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2018
Description
Biological fluids, in particular blood plasma, provide a vital source of information on the state of human health. While specific detection of biomarker species can aid in disease diagnostics, the complexity of plasma makes analysis challenging. Despite the challenge of complex sample analysis, biomarker quantification has become a primary interest in biomedical analysis. Due to the extremely specific interaction between antibody and analyte, immunoassays are attractive for the analysis of these samples and have gained popularity since their initial introduction several decades ago. Current limitations to diagnostics through blood testing include long incubation times, interference from non-specific binding, and the requirement for specialized instrumentation and personnel. Optimizing the features of immunoassay for diagnostic testing and biomarker quantification would enable early and accurate detection of disease and afford rapid intervention, potentially improving patient outcomes. Improving the limit of quantitation for immunoassay has been the primary goal of many diverse experimental platforms. While the ability to accurately quantify low abundance species in a complex biological sample is of the utmost importance in diagnostic testing, models illustrating experimental limitations have relied on mathematical fittings, which cannot be directly related to finite analytical limits or fundamental relationships. By creating models based on the law of mass action, it is demonstrated that fundamental limitations are imposed by molecular shot noise, creating a finite statistical limitation to quantitative abilities. Regardless of sample volume, 131 molecules are necessary for quantitation to take place with acceptable levels of uncertainty. Understanding the fundamental limitations of the technique can aid in the design of immunoassay platforms, and assess progress toward the development of optimal diagnostic testing. A sandwich-type immunoassay was developed and tested on three separate human protein targets: myoglobin, heart-type fatty acid binding protein, and cardiac troponin I, achieving superior limits of quantitation approaching ultimate limitations. Furthermore, this approach is compatible with upstream sample separation methods, enabling the isolation of target molecules from a complex biological sample. Isolation of target species prior to analysis allows for the multiplex detection of biomarker panels in a microscale device, making the full optimization of immunoassay techniques possible for clinical diagnostics.
ContributorsWoolley, Christine F (Author) / Hayes, Mark A. (Thesis advisor) / Ros, Alexandra (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2015
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
Detection of molecular interactions is critical for understanding many biological processes, for detecting disease biomarkers, and for screening drug candidates. Fluorescence-based approach can be problematic, especially when applied to the detection of small molecules. Various label-free techniques, such as surface plasmon resonance technique are sensitive to mass, making it extremely challenging to detect small molecules. In this thesis, novel detection methods for molecular interactions are described.
First, a simple detection paradigm based on reflectance interferometry is developed. This method is simple, low cost and can be easily applied for protein array detection.
Second, a label-free charge sensitive optical detection (CSOD) technique is developed for detecting of both large and small molecules. The technique is based on that most molecules relevant to biomedical research and applications are charged or partially charged. An optical fiber is dipped into the well of a microplate. It detects the surface charge of the fiber, which does not decrease with the size (mass) of the molecule, making it particularly attractive for studying small molecules.
Third, a method for mechanically amplification detection of molecular interactions (MADMI) is developed. It provides quantitative analysis of small molecules interaction with membrane proteins in intact cells. The interactions are monitored by detecting a mechanical deformation in the membrane induced by the molecular interactions. With this novel method small molecules and membrane proteins interaction in the intact cells can be detected. This new paradigm provides mechanical amplification of small interaction signals, allowing us to measure the binding kinetics of both large and small molecules with membrane proteins, and to analyze heterogeneous nature of the binding kinetics between different cells, and different regions of a single cell.
Last, by tracking the cell membrane edge deformation, binding caused downstream event – granule secretory has been measured. This method focuses on the plasma membrane change when granules fuse with the cell. The fusion of granules increases the plasma membrane area and thus the cell edge expands. The expansion is localized at the vesicle release location. Granule size was calculated based on measured edge expansion. The membrane deformation due to the granule release is real-time monitored by this method.
First, a simple detection paradigm based on reflectance interferometry is developed. This method is simple, low cost and can be easily applied for protein array detection.
Second, a label-free charge sensitive optical detection (CSOD) technique is developed for detecting of both large and small molecules. The technique is based on that most molecules relevant to biomedical research and applications are charged or partially charged. An optical fiber is dipped into the well of a microplate. It detects the surface charge of the fiber, which does not decrease with the size (mass) of the molecule, making it particularly attractive for studying small molecules.
Third, a method for mechanically amplification detection of molecular interactions (MADMI) is developed. It provides quantitative analysis of small molecules interaction with membrane proteins in intact cells. The interactions are monitored by detecting a mechanical deformation in the membrane induced by the molecular interactions. With this novel method small molecules and membrane proteins interaction in the intact cells can be detected. This new paradigm provides mechanical amplification of small interaction signals, allowing us to measure the binding kinetics of both large and small molecules with membrane proteins, and to analyze heterogeneous nature of the binding kinetics between different cells, and different regions of a single cell.
Last, by tracking the cell membrane edge deformation, binding caused downstream event – granule secretory has been measured. This method focuses on the plasma membrane change when granules fuse with the cell. The fusion of granules increases the plasma membrane area and thus the cell edge expands. The expansion is localized at the vesicle release location. Granule size was calculated based on measured edge expansion. The membrane deformation due to the granule release is real-time monitored by this method.
ContributorsGuan, Yan (Author) / Tao, Nongjian (Thesis advisor) / LaBaer, Joshua (Committee member) / Goryll, Michael (Committee member) / Wang, Shaopeng (Committee member) / Arizona State University (Publisher)
Created2015