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Alternative polyadenylation (APA) is the biological mechanism in which the same gene can have multiple 3'untranslated region (3'UTR) isoforms due to the presence of multiple polyadenylation signal (PAS) elements within the pre mRNAs. Because APA produces mRNA transcripts that have different 3'UTR isoforms, certain transcripts may be subject to post-transcriptional regulation by regulatory non-coding RNAs, such as microRNAs or RNA binding proteins defects of which have been implicated in diseases such as cancer. Despite the increasing level of information, functional understanding of the molecular mechanisms involved in transcription is still poorly understood, nor is it clear why APA is necessary at a cell or tissue-specific level. To address these questions I wanted to develop a set of sensor strain plasmids capable of detecting cleavage and polyadenylation in vivo, inject the complete sensor strain plasmid into C. elegans and prepare stable transgenic lines, and perform proof-of-principle RNAi feeding experiments targeting genes associated with the cleavage and polyadenylation complex machinery. I demonstrated that it was possible to create a plasmid capable of detecting cleavage and polyadenylation in C. elegans; however, issues arose during the RNAi assays indicating the sensor strain plasmid was not sensitive enough to the RNAi to effectively detect in the worms. Once the problems involved with sensitivity and variability in the RNAi effects are resolved, the plasmid would be able to better address questions regarding the functional understanding of molecular mechanisms involved in transcription termination.
ContributorsWilky, Henry Patrick (Author) / Mangone, Marco (Thesis director) / Newbern, Jason (Committee member) / Blazie, Stephen (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
The successful reduction of CO2 and protons by a light-induced cobalt porphyrin/cytb562 hybrid metalloenzyme in water is reported. Incorporation of the porphyrin into a protein scaffold results in increases in CO and H2 production over naked porphyrin. Rational point mutations to the CoPPIX binding site of cytb562 modulate production, indicating possible further improvements in catalytic activity.
ContributorsGwerder, Noah D (Author) / Ghirlanda, Giovanna (Thesis director) / Williams, Peter (Committee member) / Mangone, Marco (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Duchenne muscular dystrophy (DMD) is a lethal, X-linked disease which occurs in approximately 1 in 3,500 male births. This disease is characterized by progressive muscle wasting and causes premature death. One of the earliest symptoms of this disease is mitochondrial dysfunction. Dystrophin is a protein found under the sarcolemma. The N terminus binds to actin and the C terminus binds to dystrophin glycoprotein complex (DGC). DMD is caused by mutations in the dystrophin gene. C. elegans possess an ortholog of dystrophin, DYS-1. Though there is evidence that C. elegans can be used as a model organism to model DMD, nematode DGC has not been well characterized. Additionally, while we know that mitochondrial dysfunction has been found in humans and other model organisms, this has not been well defined in C. elegans. In order to address these issues, we crossed the SJ4103 worm strain (myo-3p::GFP(mit)) with dys-1(cx18) in order to visualize and quantify changes in mitochondria in a dys-1 background. SJ4103;cx18 nematodes were found to have less mitochondrial than SJ4103 which suggests mitochondrial dysfunction does occur in dys-1 worms. Furthermore, mitochondrial dysfunction was studied by knocking down members of the DGC, dys-1, dyb-1, sgn-1, sgca-1, and sgcb-1 in SJ4103 strain. Knock down of each gene resulted in decrease in abundance of mitochondria which suggests that each member of the DGC contributes to the overall health of nematode muscle. The ORF of dyb-1 was successfully cloned and tagged with GFP in order to visualize this DGC member C. elegans. Imaging of the transgenic dyb-1::GFP worm shows green fluoresce expressed in which suggests that dyb-1 is a functional component of the muscle fibers. This project will enable us to better understand the effects of dystrophin deficiency on mitochondrial function as well as visualize the expression of certain members of the DGC in order to establish C. elegans as a good model organism to study this disease.
ContributorsObrien, Shannon Nishino (Author) / Mangone, Marco (Thesis director) / Newbern, Jason (Committee member) / Hrach, Heather (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Cleavage and polyadenylation is a step in mRNA processing in which the 3’UTR is cleaved and a polyA tail is added to create a final mature transcript. This process relies on RNA sequence elements that guide a large multimeric protein complex named the Cleavage and Polyadenylation Complex to dock on the 3’UTR and execute the cleavage reaction. Interactions of the complex with the RNA and specific dynamics of complex recruitment and formation still remain largely uncharacterized. In our lab we have identified an Adenosine residue as the nucleotide most often present at the cleavage site, although it is unclear whether this specific element is a required instructor of cleavage and polyadenylation. To address whether the Adenosine residue is necessary and sufficient for the cleavage and polyadenylation reaction, we mutated this nucleotide at the cleavage site in three C. elegans protein coding genes, forcing the expression of these wt and mutant 3’UTRs, and studied how the cleavage and polyadenylation machinery process these genes in vivo. We found that interrupting the wt sequence elements found at the cleavage site interferes with the cleavage and polyadenylation reaction, suggesting that the sequence close to the end of the transcript plays a role in modulating the site of the RNA cleavage. This activity is also gene-specific. Genes such as ges-1 showed little disruption in the cleavage of the transcript, with similar location occurring in both the wt and mutant 3’UTRs. On the other hand, mutation of the cleavage site in genes such as Y106G6H.9 caused the activation of new cryptic cleavage sites within the transcript. Taken together, my experiments suggest that the sequence elements at the cleavage site somehow participate in the reaction to guide the cleavage reaction to occur at an exact site. This work will help to better understand the mechanisms of transcription termination in vivo and will push forward research aimed to study post-transcriptional gene regulation in eukaryotes.
ContributorsSteber, Hannah Suzanne (Author) / Mangone, Marco (Thesis director) / Harris, Robin (Committee member) / LaBaer, Joshua (Committee member) / School of Life Sciences (Contributor, Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Nucleic acid polymers have numerous applications in both therapeutics and research to control gene expression and bind biologically relevant targets. However, due to poor biological stability their clinical applications are limited. Chemical modifications can improve both intracellular and extracellular stability and enhance resistance to nuclease degradation. To identify a potential candidate for a highly stable synthetic nucleic acid, the biostability of α-L-threofuranosyl nucleic acid (TNA) was evaluated under simulated biological conditions. TNA contains a four-carbon sugar and is linked by 2’, 3’ phosphodiester bonds. We hypothesized that this distinct chemical structure would yield greater nuclease resistance in human serum and human liver microsomes, which were selected as biologically relevant nuclease conditions. We found that TNA oligonucleotides remained undigested for 7 days in these conditions. In addition, TNA/DNA heteropolymers and TNA/RNA oligonucleotide duplexes displayed nuclease resistance, suggesting that TNA has a protective effect over DNA and RNA. In conclusion TNA demonstrates potential as a viable synthetic nucleic acid for use in numerous clinical and therapeutic applications.
ContributorsCulbertson, Michelle Catherine (Author) / Maley, Carlo (Thesis director) / Mangone, Marco (Committee member) / Larsen, Andrew (Committee member) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Obesity and related health disparities including type 2 diabetes disproportionately impact Latino youth. These health disparities may be the result of gene-environment interactions, but limited research has examined these interactions in the pediatric age group. Lifestyle intervention is the cornerstone for preventing diabetes among high-risk populations and epigenetic and genetic factors may help explain the biological mechanisms underlying diabetes risk reduction following lifestyle changes. MicroRNAs (miRNAs) are small, non-coding RNA’s that regulate gene expression and have emerged as potential biomarkers for predicting type 2 diabetes risk in adults but have yet to be applied to youth. Therefore, the purpose of this study was to identify changes in miRNA expression among Latino youth with prediabetes (4 female/2 male, ages 14-16, BMI percentile 99 ±.2) who participated in a 12-week lifestyle intervention focused on increasing physical activity and improving nutrition-related behaviors.
ContributorsKarch, Jamie (Co-author) / Day, Samantha (Co-author) / Shaibi, Gabriel (Thesis director) / Coletta, Dawn (Committee member) / Arizona State University. College of Nursing & Healthcare Innovation (Contributor) / College of Integrative Sciences and Arts (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Disturbances in the protein interactome often play a large role in cancer progression. Investigation of protein-protein interactions (PPI) can increase our understanding of cancer pathways and will disclose unknown targets involved in cancer disease biology. Although numerous methods are available to study protein interactions, most platforms suffer from drawbacks including high false positive rates, low throughput, and lack of quantification. Moreover, most methods are not compatible for use in a clinical setting. To address these limitations, we have developed a multiplexed, in-solution protein microarray (MISPA) platform with broad applications in proteomics. MISPA can be used to quantitatively profile PPIs and as a robust technology for early detection of cancers. This method utilizes unique DNA barcoding of individual proteins coupled with next generation sequencing to quantitatively assess interactions via barcode enrichment. We have tested the feasibility of this technology in the detection of patient immune responses to oropharyngeal carcinomas and in the discovery of novel PPIs in the B-cell receptor (BCR) pathway. To achieve this goal, 96 human papillomavirus (HPV) antigen genes were cloned into pJFT7-cHalo (99% success) and pJFT7-n3xFlag-Halo (100% success) expression vectors. These libraries were expressed via a cell-free in vitro transcription-translation system with 93% and 96% success, respectively. A small-scale study of patient serum interactions with barcoded HPV16 antigens was performed and a HPV proteome-wide study will follow using additional patient samples. In addition, 15 query proteins were cloned into pJFT7_nGST expression vectors, expressed, and purified with 93% success to probe a library of 100 BCR pathway proteins and detect novel PPIs.
ContributorsRinaldi, Capria Lakshmi (Author) / LaBaer, Joshua (Thesis director) / Mangone, Marco (Committee member) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
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
Traditional Reinforcement Learning (RL) assumes to learn policies with respect to reward available from the environment but sometimes learning in a complex domain requires wisdom which comes from a wide range of experience. In behavior based robotics, it is observed that a complex behavior can be described by a combination of simpler behaviors. It is tempting to apply similar idea such that simpler behaviors can be combined in a meaningful way to tailor the complex combination. Such an approach would enable faster learning and modular design of behaviors. Complex behaviors can be combined with other behaviors to create even more advanced behaviors resulting in a rich set of possibilities. Similar to RL, combined behavior can keep evolving by interacting with the environment. The requirement of this method is to specify a reasonable set of simple behaviors. In this research, I present an algorithm that aims at combining behavior such that the resulting behavior has characteristics of each individual behavior. This approach has been inspired by behavior based robotics, such as the subsumption architecture and motor schema-based design. The combination algorithm outputs n weights to combine behaviors linearly. The weights are state dependent and change dynamically at every step in an episode. This idea is tested on discrete and continuous environments like OpenAI’s “Lunar Lander” and “Biped Walker”. Results are compared with related domains like Multi-objective RL, Hierarchical RL, Transfer learning, and basic RL. It is observed that the combination of behaviors is a novel way of learning which helps the agent achieve required characteristics. A combination is learned for a given state and so the agent is able to learn faster in an efficient manner compared to other similar approaches. Agent beautifully demonstrates characteristics of multiple behaviors which helps the agent to learn and adapt to the environment. Future directions are also suggested as possible extensions to this research.
ContributorsVora, Kevin Jatin (Author) / Zhang, Yu (Thesis advisor) / Yang, Yezhou (Committee member) / Praharaj, Sarbeswar (Committee member) / Arizona State University (Publisher)
Created2021
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