Matching Items (114)
Description
The splicing of precursor messenger RNAs (pre-mRNAs) plays an essential role in dictating the mature mRNA profiles of eukaryotic cells. Mis-regulation of splicing, due to mutations in pre-mRNAs or in components of the splicing machinery, is associated with many diseases. Therefore, knowledge of pre-mRNA splicing mechanisms is required to understand gene expression regulation during states of homeostasis and disease, and for the development of therapeutic interventions.Splicing is catalyzed by the spliceosome, a dynamic and protein-rich ribozyme composed of five small nuclear ribonucleoproteins (snRNPs) and ~170 auxiliary factors. Early interactions that occur in prespliceosomal complexes formed by the 5′- and 3′-splice-site bound U1 and U2 snRNPs are responsible for committing introns for removal. However, the mechanisms underlying these early interactions remain to be fully characterized for understanding the influence of alternative splicing factors and the impact of recurrent disease-associated mutations in prespliceosomal proteins. The goal of my dissertation research was to delineate the role of the U1 small nuclear RNA (snRNA) during prespliceosome assembly. By applying a cellular minigene reporter assay and a variety of in vitro techniques including cell-free protein expression, UV-crosslinking, electrophoretic mobility shift assays, surface plasmon resonance, and RNA affinity purification, my work establishes critical roles for the U1 snRNA stem-loops 3 (SL3) and 4 (SL4) in formation of intron definition interactions during prespliceosome assembly. Previously, the SL4 of the U1 snRNA was shown to form a molecular bridge across introns by contacting the U2-specific splicing factor 3A1 (SF3A1). I identified the Ubiquitin-like domain of SF3A1 as a non-canonical RNA binding domain responsible for U1-SL4 binding. I also determined a role for the SL3 region of the U1 snRNA in splicing and characterized the spliceosomal RNA helicase UAP56 as an SL3 interacting protein. By knocking-down the SL3- and SL4-interacting proteins, I confirmed that U1 splicing activity in vivo relies on UAP56 and SF3A1 and that their functions are interdependent. These findings, in addition to the observations made using in vitro splicing assays, support a model whereby UAP56, through its interaction with U1-SL3, enhances the cross-intron interaction between U1-SL4 and SF3A1 to promote prespliceosome formation.
ContributorsMartelly, William (Author) / Sharma, Shalini (Thesis advisor) / Mangone, Marco (Thesis advisor) / Gustin, Kurt (Committee member) / Chen, Julian (Committee member) / Arizona State University (Publisher)
Created2021
Description
The oceanic biological carbon pump is a key component of the global carbon cycle in which dissolved carbon dioxide is taken up by phytoplankton during photosynthesis, a fraction of which then sinks to depth and contributes to oceanic carbon storage. The small-celled phytoplankton (<5 µm) that dominate the phytoplankton community in oligotrophic oceans have traditionally been viewed as contributing little to export production due to their small size. However, recent studies have shown that the picocyanobacterium Synechococcus produces transparent exopolymer particles (TEP), the sticky matrix of marine aggregates, and forms abundant microaggregates (5-60 µm), which is enhanced under nutrient limited growth conditions. Whether other small phytoplankton species exude TEP and form microaggregates, and if these are enhanced under growth-limiting conditions remains to be investigated. This study aims to analyze how nutrient limitation affects TEP production and microaggregate formation of species that are found to be associated with sinking particles in the Sargasso Sea. The pico-cyanobacterium Prochlorococcus marinus (0.8 µm), the nano-diatom Minutocellus polymorphus (2 µm), and the pico-prasinophyte Ostreococcus lucimarinus (0.6 µm) were grown in axenic batch culture experiments under nutrient replete and limited conditions. It was hypothesized that phytoplankton subject to nutrient limitation will aggregate more than those under replete conditions due to an increased exudation of TEP and that Minutocellus would produce the most TEP and microaggregates while Prochlorococcus would produce the least TEP and microaggregates of the three phytoplankton groups. As hypothesized, nutrient limitation increased TEP concentration in all three species, however they were only significant in nitrogen-limited treatments of Prochlorococcus as well as nitrogen- and phosphorus-limited treatments of Minutocellus. Formation of microaggregates was significantly enhanced in Minutocellus and Ostreococcus cultures in distinct microaggregate size ranges. Minutocellus produced the most TEP per cell and aggregated at higher volume concentrations compared to Prochlorococcus and Ostreococcus. Surprisingly, Ostreococcus produced more TEP than Prochlorococcus and Minutocellus per unit cell volume. These findings show for the first time how nutrient limited conditions enhance TEP production and microaggregation of Prochlorococcus, Minutocellus, and Ostreococcus, providing a mechanism for their incorporation into larger, sinking particles and contribution to export production in oligotrophic oceans.
ContributorsShurtleff, Catrina (Author) / Neuer, Susanne (Thesis advisor) / Lomas, Michael W. (Committee member) / Garcia-Pichel, Ferran (Committee member) / Arizona State University (Publisher)
Created2022
Description
Under current climate conditions northern peatlands mostly act as C sinks; however, changes in climate and environmental conditions, can change the soil carbon decomposition cascade, thus altering the sink status. Here I studied one of the most abundant northern peatland types, poor fen, situated along a climate gradient from tundra (Daring Lake, Canada) to boreal forest (Lutose, Canada) to temperate broadleaf and mixed forest (Bog Lake, MN and Chicago Bog, NY) biomes to assess patterns of microbial abundance across the climate gradient. Principal component regression analysis of the microbial community and environmental variables determined that mean annual temperature (MAT) (r2=0.85), mean annual precipitation (MAP) (r2=0.88), and soil temperature (r2=0.77), were the top significant drivers of microbial community composition (p < 0.001). Niche breadth analysis revealed the relative abundance of Intrasporangiaceae, Methanobacteriaceae and Candidatus Methanoflorentaceae fam. nov. to increase when MAT and MAP decrease. The same analysis showed Spirochaetaceae, Methanosaetaceae and Methanoregulaceae to increase in relative abundance when MAP, soil temperature and MAT increased, respectively. These findings indicated that climate variables were the strongest predictors of microbial community composition and that certain taxa, especially methanogenic families demonstrate distinct patterns across the climate gradient. To evaluate microbial production of methanogenic substrates, I carried out High Resolution-DNA-Stable Isotope Probing (HR-DNA-SIP) to evaluate the active portion of the community’s intermediary ecosystem metabolic processes. HR-DNA-SIP revealed several challenges in efficiency of labelling and statistical identification of responders, however families like Veillonellaceae, Magnetospirillaceae, Acidobacteriaceae 1, were found ubiquitously active in glucose amended incubations. Differences in metabolic byproducts from glucose amendments show distinct patterns in acetate and propionate accumulation across sites. Families like Spirochaetaceae and Sphingomonadaceae were only found to be active in select sites of propionate amended incubations. By-product analysis from propionate incubations indicate that the northernmost sites were acetate-accumulating communities.
These results indicate that microbial communities found in poor fen northern peatlands are strongly influenced by climate variables predicted to change under current climate scenarios. I have identified patterns of relative abundance and activity of select microbial taxa, indicating the potential for climate variables to influence the metabolic pathway in which carbon moves through peatland systems.
ContributorsSarno, Analissa Flores (Author) / Cadillo-Quiroz, Hinsby (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Krajmalnik-Brown, Rosa (Committee member) / Childers, Daniel (Committee member) / Arizona State University (Publisher)
Created2022
Description
The ability to find evidence of life on early Earth and other planets is constrained by the current understanding of biosignatures and our ability to differentiate fossils from abiotic mimics. When organisms transition from the living realm to the fossil record, their morphological and chemical characteristics are modified, usually resulting in the loss of information. These modifications can happen during early and late diagenesis and differ depending on local geochemical properties. These post-depositional modifications need to be understood to better interpret the fossil record. Siliceous hot spring deposits (sinters) are of particular interest for biosignature research as they are early Earth analog environments and targets for investigating the presence of fossil life on Mars. As silica-supersaturated fluids flow from the vent to the distal apron, they precipitate non-crystalline opal-A that fossilizes microbial communities at a range in scales (μm-cm). Therefore, many studies have documented the ties between the active microbial communities and the morphological and chemical biosignatures in hot springs. However, far less attention has been placed on understanding preservation in systems with complex mineralogy or how post-depositional alteration affects the retention of biosignatures. Without this context, it can be challenging to recognize biosignatures in ancient rocks. This dissertation research aims to refine our current understanding of biosignature preservation and retention in sinters. Biosignatures of interest include organic matter, microfossils, and biofabrics. The complex nature of hot springs requires a comprehensive understanding of biosignature preservation that is representative of variable chemistries and post-depositional alterations. For this reason, this dissertation research chapters are field site-based. Chapter 2 investigates biosignature preservation in an unusual spring with mixed opal-A-calcite mineralogy at Lýsuhóll, Iceland. Chapter 3 tracks how silica diagenesis modifies microfossil morphology and associated organic matter at Puchuldiza, Chile. Chapter 4 studies the effects of acid fumarolic overprinting on biosignatures in Gunnuhver, Iceland. To accomplish this, traditional geologic methods (mapping, petrography, X-ray diffraction, bulk elemental analyses) were combined with high-spatial-resolution elemental mapping to better understand diagenetic effects in these systems. Preservation models were developed to predict the types and styles of biosignatures that can be present depending on the depositional and geochemical context. Recommendations are also made for the types of deposits that are most likely to preserve biosignatures.
ContributorsJuarez Rivera, Marisol (Author) / Farmer, Jack D (Thesis advisor) / Hartnett, Hilairy E (Committee member) / Shock, Everett (Committee member) / Garcia-Pichel, Ferran (Committee member) / Trembath-Reichert, Elizabeth (Committee member) / Arizona State University (Publisher)
Created2021
Description
Predatory bacteria are a guild of heterotrophs that feed directly on other living bacteria. They belong to several bacterial lineages that evolved this mode of life independently and occur in many microbiomes and environments. Current knowledge of predatory bacteria is based on culture studies and simple detection in natural systems. The ecological consequences of their activity, unlike those of other populational loss factors like viral infection or grazing by protists, are yet to be assessed. During large-scale cultivation of biological soil crusts intended for arid soil rehabilitation, episodes of catastrophic failure were observed in cyanobacterial growth that could be ascribed to the action of an unknown predatory bacterium using bioassays. This predatory bacterium was also present in natural biocrust communities, where it formed clearings (plaques) up to 9 cm in diameter that were visible to the naked eye. Enrichment cultivation and purification by cell-sorting were used to obtain co-cultures of the predator with its cyanobacterial prey, as well as to identify and characterize it genomically, physiologically and ultrastructurally. A Bacteroidetes bacterium, unrelated to any known isolate at the family level, it is endobiotic, non-motile, obligately predatory, displays a complex life cycle and very unusual ultrastructure. Extracellular propagules are small (0.8-1.0 µm) Gram-negative cocci with internal two-membrane-bound compartmentalization. These gain entry to the prey likely using a suite of hydrolytic enzymes, localizing to the cyanobacterial cytoplasm, where growth begins into non-compartmentalized pseudofilaments that undergo secretion of vesicles and simultaneous multiple division to yield new propagules. I formally describe it as Candidatus Cyanoraptor togatus, hereafter Cyanoraptor. Its prey range is restricted to biocrust-forming, filamentous, non-heterocystous, gliding, bundle-making cyanobacteria. Molecular meta-analyses showed its worldwide distribution in biocrusts. Biogeochemical analyses of Cyanoraptor plaques revealed that it causes a complete loss of primary productivity, and significant decreases in other biocrusts properties such as water-retention and dust-trapping capacity. Extensive field surveys in the US Southwest revealed its ubiquity and its dispersal-limited, aggregated spatial distribution and incidence. Overall, its activity reduces biocrust productivity by 10% at the ecosystem scale. My research points to predatory bacteria as a significant, but overlooked, ecological force in shaping soil microbiomes.
ContributorsBethany Rakes, Julie Ann (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Gile, Gillian (Committee member) / Cao, Huansheng (Committee member) / Jacobs, Bertram (Committee member) / Arizona State University (Publisher)
Created2022
Description
Glioblastoma (GBM), the most common and aggressive primary brain tumor affecting adults, is characterized by an aberrant yet druggable epigenetic landscape. The Histone Deacetylases (HDACs), a major family of epigenetic regulators, favor transcriptional repression by mediating chromatin compaction and are frequently overexpressed in human cancers, including GBM. Hence, over the last decade there has been considerable interest in using HDAC inhibitors (HDACi) for the treatment of malignant primary brain tumors. However, to date most HDACi tested in clinical trials have failed to provide significant therapeutic benefit to patients with GBM. This is because current HDACi have poor or unknown pharmacokinetic profiles, lack selectivity towards the different HDAC isoforms, and have narrow therapeutic windows. Isoform selectivity for HDACi is important given that broad inhibition of all HDACs results in widespread toxicity across different organs. Moreover, the functional roles of individual HDAC isoforms in GBM are still not well understood. Here, I demonstrate that HDAC1 expression increases with brain tumor grade and is correlated with decreased survival in GBM. I find that HDAC1 is the essential HDAC isoform in glioma stem cells and its loss is not compensated for by its paralogue HDAC2 or other members of the HDAC family. Loss of HDAC1 alone has profound effects on the glioma stem cell phenotype in a p53-dependent manner and leads to significant suppression of tumor growth in vivo. While no HDAC isoform-selective inhibitors are currently available, the second-generation HDACi quisinostat harbors high specificity for HDAC1. I show that quisinostat exhibits potent growth inhibition in multiple patient-derived glioma stem cells. Using a pharmacokinetics- and pharmacodynamics-driven approach, I demonstrate that quisinostat is a brain-penetrant molecule that reduces tumor burden in flank and orthotopic models of GBM and significantly extends survival both alone and in combination with radiotherapy. The work presented in this thesis thereby unveils the non-redundant functions of HDAC1 in therapy- resistant glioma stem cells and identifies a brain-penetrant HDACi with higher selectivity towards HDAC1 as a potent radiosensitizer in preclinical models of GBM. Together, these results provide a rationale for developing quisinostat as a potential adjuvant therapy for the treatment of GBM.
ContributorsLo Cascio, Costanza (Author) / LaBaer, Joshua (Thesis advisor) / Mehta, Shwetal (Committee member) / Mirzadeh, Zaman (Committee member) / Mangone, Marco (Committee member) / Paek, Andrew (Committee member) / Arizona State University (Publisher)
Created2022
Description
Mycobacterial infections, as represented by leprosy and tuberculosis, have persisted as human pathogens for millennia. Their environmental counterparts, nontuberculous mycobacteria (NTM), are commodious infectious agents endowed with extensive innate and acquired antimicrobial resistance. The current drug development process selects for antibiotics with high specificity for definitive targets within bacterial metabolic and replication pathways. Because these compounds demonstrate limited efficacy against mycobacteria, novel antimycobacterial agents with unconventional mechanisms of action were identified. Two highly resistant NTMs, Mycobacterium abscessus (Mabs) a rapid-growing respiratory, skin, and soft tissue pathogen, and Mycobacterium ulcerans (MU), the causative agent of Buruli ulcer, were selected as targets. Compounds that indicated antimicrobial activity against other highly resistant pathogens were selected for initial screening. Antimicrobial peptides (AMPs) have demonstrated activity against a variety of bacterial pathogens, including mycobacterial species. Designed antimicrobial peptides (dAMPs), rationally-designed and synthetic contingents, combine iterative features of natural AMPs to achieve superior antimicrobial activity in resistant pathogens. Initial screening identified two dAMPs, RP554 and RP557, with bactericidal activity against Mabs. Clay-associated ions have previously demonstrated bactericidal activity against MU. Synthetic and customizable aluminosilicates have also demonstrated adsorption of bacterial cells and toxins. On this basis, two aluminosilicate materials, geopolymers (GP) and ion-exchange nanozeolites (IE-nZeos), were screened for antimicrobial activity against MU and its fast-growing relative, Mycobacterium marinum (Mmar). GPs demonstrated adsorption of MU cells and mycolactone, a secreted, lipophilic toxin, whereas Cu-nZeos and Ag-nZeos demonstrated antibacterial activity against MU and Mmar. Cumulatively, these results indicate that an integrative drug selection process may yield a new generation of antimycobacterial agents.
ContributorsDermody, Roslyn June (Author) / Haydel, Shelley E (Thesis advisor) / Bean, Heather (Committee member) / Nickerson, Cheryl (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2022
Description
Advances in sequencing technology have generated an enormous amount of data over the past decade. Equally advanced computational methods are needed to conduct comparative and functional genomic studies on these datasets, in particular tools that appropriately interpret indels within an evolutionary framework. The evolutionary history of indels is complex and often involves repetitive genomic regions, which makes identification, alignment, and annotation difficult. While previous studies have found that indel lengths in both deoxyribonucleic acid and proteins obey a power law, probabilistic models for indel evolution have rarely been explored due to their computational complexity. In my research, I first explore an application of an expectation-maximization algorithm for maximum-likelihood training of a codon substitution model. I demonstrate the training accuracy of the expectation-maximization on my substitution model. Then I apply this algorithm on a published 90 pairwise species dataset and find a negative correlation between the branch length and non-synonymous selection coefficient. Second, I develop a post-alignment fixation method to profile each indel event into three different phases according to its codon position. Because current codon-aware models can only identify the indels by placing the gaps between codons and lead to the misalignment of the sequences. I find that the mouse-rat species pair is under purifying selection by looking at the proportion difference of the indel phases. I also demonstrate the power of my sliding-window method by comparing the post-aligned and original gap positions. Third, I create an indel-phase moore machine including the indel rates of three phases, length distributions, and codon substitution models. Then I design a gillespie simulation that is capable of generating true sequence alignments. Next I develop an importance sampling method within the expectation-maximization algorithm that can successfully train the indel-phase model and infer accurate parameter estimates from alignments. Finally, I extend the indel phase analysis to the 90 pairwise species dataset across three alignment methods, including Mafft+sw method developed in chapter 3, coati-sampling methods applied in chapter 4, and coati-max method. Also I explore a non-linear relationship between the dN/dS and Zn/(Zn+Zs) ratio across 90 species pairs.
ContributorsZhu, Ziqi (Author) / Cartwright, Reed A (Thesis advisor) / Taylor, Jay (Committee member) / Wideman, Jeremy (Committee member) / Mangone, Marco (Committee member) / Arizona State University (Publisher)
Created2022
Description
Protein-nucleic acid interactions are ubiquitous in biological systems playing a pivotal role in fundamental processes such as replication, transcription and translation. These interactions have been extensively used to develop biosensors, imaging techniques and diagnostic tools.This dissertation focuses on design of a small molecule responsive biosensor that employs transcription factor/deoxyribonucleic acid (DNA) interactions to detect 10 different analytes including antibiotics such as tetracyclines and erythromycin. The biosensor harnesses the multi-turnover collateral cleavage activity of Cas12a to provide signal amplification in less than an hour that can be monitored using fluorescence as well as on paper based diagnostic devices. In addition, the functionality of this assay was preserved when testing tap water and wastewater spiked with doxycycline. Overall, this biosensor has potential to expand the range of small molecule detection and can be used to identify environmental contaminants.
In second part of the dissertation, interactions between nonribosomal peptide synthetases (NRPS) and ribonucleic acid (RNA) were utilized for programming the synthesis of nonribosomal peptides. RNA scaffolds harboring peptide binding aptamers and interconnected using kissing loops to guide the assembly of NRPS modules modified with corresponding aptamer-binding peptides were built. A successful chimeric assembly of Ent synthetase modules was shown that was characterized by the production of Enterobactin siderophore. It was found that the programmed RNA/NRPS assembly could achieve up to 60% of the yield of wild-type biosynthetic pathway of the iron-chelator enterobactin.
Finally, a cas12a-based detection method for discriminating short tandem repeats where a toehold exchange mechanism was designed to distinguish different numbers of repeats found in Huntington’s disease, Spinocerebellar ataxia type 10 and type 36. It was observed that the system discriminates well when lesser number of repeats are present and provides weaker resolution as the size of DNA strands increases. Additionally, the system can identify Kelch13 mutations such as P553L, N458Y and F446I from the wildtype sequence for Artemisinin resistance detection.
This dissertation demonstrates the great utility of harnessing protein-nucleic acid interactions to construct biomolecular devices for detecting clinically relevant nucleic acid mutations, a variety of small molecule analyte and programming the production of useful molecules.
ContributorsChaudhary, Soma (Author) / Green, Alexander (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Mangone, Marco (Committee member) / Arizona State University (Publisher)
Created2022
Description
The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic, declared in March 2020 resulted in an unprecedented scientific effort that led to the deployment in less than a year of several vaccines to prevent severe disease, hospitalizations, and death from coronavirus disease 2019 (COVID-19). Most vaccine models focus on the production of neutralizing antibodies against the spike (S) to prevent infection. As the virus evolves, new variants emerge that evade neutralizing antibodies produced by natural infection and vaccination, while memory T cell responses are long-lasting and resilient to most of the changes found in variants of concern (VOC). Several lines of evidence support the study of T cell-mediated immunity in SARS-CoV-2 infections. First, T cell reactivity against SARS-CoV-2 is found in both (cluster of differentiation) CD4+ and CD8+ T cell compartments in asymptomatic, mild, and severe recovered COVID-19 patients. Second, an early and stronger CD8+ T cell response correlates with less severe COVID-19 disease [1-4]. Third, both CD4+ and CD8+ T cells that are reactive to SARS-CoV-2 viral antigens are found in healthy unexposed individuals suggesting that cross-reactive and conserved epitopes may be protective against infection.
The current study is focused on the T cell-mediated response, with special attention to conserved, non-spike-cross-reactive epitopes that may be protective against SARS-CoV-2. The first chapter reviews the importance of epitope prediction in understanding the T cell-mediated responses to a pathogen. The second chapter centers on the validation of SARS-CoV-2 CD8+ T cell predicted peptides to find conserved, immunodominant, and immunoprevalent epitopes that can be incorporated into the next generation of vaccines against severe COVID-19 disease. The third chapter explores pre-existing immunity to SARS-CoV-2 in a pre-pandemic cohort and finds two highly immunogenic epitopes that are conserved among human common cold coronaviruses (HCoVs). To end, the fourth chapter explores the concept of T cell receptor (TCR) cross-reactivity by isolating SARS-CoV-2-reactive TCRs to elucidate the mechanisms of cross-reactivity to SARS-CoV-2 and other human coronaviruses (HCoVs).
ContributorsCarmona, Jacqueline (Author) / Anderson, Karen S (Thesis advisor) / Lake, Douglas (Thesis advisor) / Maley, Carlo (Committee member) / Mangone, Marco (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2023