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- All Subjects: Biochemistry
- Genre: Academic theses
- Status: Published
Description
Telomerase enzyme is a truly remarkable enzyme specialized for the addition of short, highly repetitive DNA sequences onto linear eukaryotic chromosome ends. The telomerase enzyme functions as a ribonucleoprotein, minimally composed of the highly conserved catalytic telomerase reverse transcriptase and essential telomerase RNA component containing an internalized short template region within the vastly larger non-coding RNA. Even among closely related groups of species, telomerase RNA is astonishingly divergent in sequence, length, and secondary structure. This massive disparity is highly prohibitive for telomerase RNA identification from previously unexplored groups of species, which is fundamental for secondary structure determination. Combined biochemical enrichment and computational screening methods were employed for the discovery of numerous telomerase RNAs from the poorly characterized echinoderm lineage. This resulted in the revelation that--while closely related to the vertebrate lineage and grossly resembling vertebrate telomerase RNA--the echinoderm telomerase RNA central domain varies extensively in structure and sequence, diverging even within echinoderms amongst sea urchins and brittle stars. Furthermore, the origins of telomerase RNA within the eukaryotic lineage have remained a persistent mystery. The ancient Trypanosoma telomerase RNA was previously identified, however, a functionally verified secondary structure remained elusive. Synthetic Trypanosoma telomerase was generated for molecular dissection of Trypanosoma telomerase RNA revealing two RNA domains functionally equivalent to those found in known telomerase RNAs, yet structurally distinct. This work demonstrates that telomerase RNA is uncommonly divergent in gross architecture, while retaining critical universal elements.
ContributorsPodlevsky, Joshua (Author) / Chen, Julian (Thesis advisor) / Mangone, Marco (Committee member) / Kusumi, Kenro (Committee member) / Wilson-Rawls, Norma (Committee member) / Arizona State University (Publisher)
Created2015
Description
Advances in chemical synthesis have enabled new lines of research with unnatural genetic polymers whose modified bases or sugar-phosphate backbones have potential therapeutic and biotechnological applications. Maximizing the potential of these synthetic genetic systems requires inventing new molecular biology tools that can both generate and faithfully replicate unnatural polymers of significant length. Threose nucleic acid (TNA) has received significant attention as a complete replication system has been developed by engineering natural polymerases to broaden their substrate specificity. The system, however, suffers from a high mutational load reducing its utility. This thesis will cover the development of two new polymerases capable of transcribing and reverse transcribing TNA polymers with high efficiency and fidelity. The polymerases are identified using a new strategy wherein gain-of-function mutations are sampled in homologous protein architectures leading to subtle optimization of protein function. The new replication system has a fidelity that supports the propagation of genetic information enabling in vitro selection of functional TNA molecules. TNA aptamers to human alpha-thrombin are identified and demonstrated to have superior stability compared to DNA and RNA in biologically relevant conditions. This is the first demonstration that functional TNA molecules have potential in biotechnology and molecular medicine.
ContributorsDunn, Matthew Ryan (Author) / Chaput, John C (Thesis advisor) / LaBaer, Joshua (Committee member) / Lake, Douglas (Committee member) / Mangone, Marco (Committee member) / Arizona State University (Publisher)
Created2015
Description
Extracellular vesicles (EVs) represent a heterogeneous population of small vesicles, consisting of a phospholipidic bilayer surrounding a soluble interior cargo. These vesicles play an important role in cellular communication by virtue of their protein, RNA, and lipid content, which can be transferred among cells. Peripheral blood is a rich source of circulating EVs. An analysis of EVs in peripheral blood could provide access to unparalleled amounts of biomarkers of great diagnostic, prognostic as well as therapeutic value. In the current study, a plasma EV enrichment method based on pluronic co-polymer was first established and characterized. Plasma EVs from breast cancer patients were then enriched, profiled and compared to non-cancer controls. Proteins signatures that contributed to the prediction of cancer samples from non-cancer controls were created by a random-forest based cross-validation approach. We found that a large portion of these signatures were related to breast cancer aggression. To verify such findings, KIAA0100, one of the features identified, was chosen for in vitro molecular and cellular studies in the breast cancer cell line MDA-MB-231. We found that KIAA0100 regulates cancer cell aggression in MDA-MB-231 in an anchorage-independent manner and is particularly associated with anoikis resistance through its interaction with HSPA1A. Lastly, plasma EVs contain not only individual proteins, but also numerous molecular complexes. In order to measure millions of proteins, isoforms, and complexes simultaneously, Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT) platform was applied. ADAPT employs an enriched library of single-stranded oligodeoxynucleotides to profile complex biological samples, thus achieving a deep coverage of system-wide, native biomolecules. Profiling of EVs from breast cancer patients was able to obtain a prediction AUC performance of 0.73 when compared biopsy-positive cancer patient to healthy controls and 0.64 compared to biopsy-negative controls and such performance was not associated with the physical breast condition indicated by BIRAD scores. Taken together, current research demonstrated the potential of profiling plasma EVs in searching for therapeutic targets as well as diagnostic signatures.
ContributorsZhong, Zhenyu (Author) / Spetzler, David (Thesis advisor) / Yan, Hao (Thesis advisor) / Lake, Douglas (Committee member) / Mangone, Marco (Committee member) / Arizona State University (Publisher)
Created2018
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
The fundamental building blocks for constructing complex synthetic gene networks are effective biological parts with wide dynamic range, low crosstalk, and modularity. RNA-based components are promising sources of such parts since they can provide regulation at the level of transcription and translation and their predictable base pairing properties enable large libraries to be generated through in silico design. This dissertation studies two different approaches for initiating interactions between RNA molecules to implement RNA-based components that achieve translational regulation. First, single-stranded domains known as toeholds were employed for detection of the highly prevalent foodborne pathogen norovirus. Toehold switch riboregulators activated by trigger RNAs from the norovirus RNA genome are designed, validated, and coupled with paper-based cell-free transcription-translation systems. Integration of paper-based reactions with synbody enrichment and isothermal RNA amplification enables as few as 160 copies/mL of norovirus from clinical samples to be detected in reactions that do not require sophisticated equipment and can be read directly by eye. Second, a new type of riboregulator that initiates RNA-RNA interactions through the loop portions of RNA stem-loop structures was developed. These loop-initiated RNA activators (LIRAs) provide multiple advantages compared to toehold-based riboregulators, exhibiting ultralow signal leakage in vivo, lacking any trigger RNA sequence constraints, and appending no additional residues to the output protein. Harnessing LIRAs as modular parts, logic gates that exploit loop-mediated control of mRNA folding state to implement AND and OR operations with up to three sequence-independent input RNAs were constructed. LIRA circuits can also be ported to paper-based cell-free reactions to implement portable systems with molecular computing and sensing capabilities. LIRAs can detect RNAs from a variety of different pathogens, such as HIV, Zika, dengue, yellow fever, and norovirus, and after coupling to isothermal amplification reactions, provide visible test results down to concentrations of 20 aM (12 RNA copies/µL). And the logic functionality of LIRA circuits can be used to specifically identify different HIV strains and influenza A subtypes. These findings demonstrate that toehold- and loop-mediated RNA-RNA interactions are both powerful strategies for implementing RNA-based computing systems for intracellular and diagnostic applications.
ContributorsMA, DUO (Author) / Green, Alexander (Thesis advisor) / Mangone, Marco (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2019
Description
Mutation is the source of heritable variation of genotype and phenotype, on which selection may act. Mutation rates describe a fundamental parameter of living things, which influence the rate at which evolution may occur, from viral pathogens to human crops and even to aging cells and the emergence of cancer. An understanding of the variables which impact mutation rates and their estimation is necessary to place mutation rate estimates in their proper contexts. To better understand mutation rate estimates, this research investigates the impact of temperature upon transcription rate error estimates; the impact of growing cells in liquid culture vs. on agar plates; the impact of many in vitro variables upon the estimation of deoxyribonucleic acid (DNA) mutation rates from a single sample; and the mutational hazard induced by expressing clustered regularly interspaced short palindromic repeat (CRISPR) proteins in yeast. This research finds that many of the variables tested did not significantly alter the estimation of mutation rates, strengthening the claims of previous mutation rate estimates across the tree of life by diverse experimental approaches. However, it is clear that sonication is a mutagen of DNA, part of an effort which has reduced the sequencing error rate of circle-seq by over 1,000-fold. This research also demonstrates that growth in liquid culture modestly skews the mutation spectrum of MMR- Escherichia coli, though it does not significantly impact the overall mutation rate. Finally, this research demonstrates a modest mutational hazard of expressing Cas9 and similar CRISPR proteins in yeast cells at an un-targeted genomic locus, though it is possible the indel rate has been increased by an order of magnitude.
ContributorsBaehr, Stephan (Author) / Lynch, Michael (Thesis advisor) / Geiler-Samerotte, Kerry (Committee member) / Mangone, Marco (Committee member) / Wilson, Melissa (Committee member) / Arizona State University (Publisher)
Created2023