Matching Items (223)
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
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
The RASopathies are a collection of developmental diseases caused by germline mutations in components of the RAS/MAPK signaling pathway and is one of the world’s most common set of genetic diseases. A majority of these mutations result in an upregulation of RAS/MAPK signaling and cause a variety of both physical and neurological symptoms. Neurodevelopmental symptoms of the RASopathies include cognitive and motor delays, learning and intellectual disabilities, and various behavioral problems. Recent noninvasive imaging studies have detected widespread abnormalities within white matter tracts in the brains of RASopathy patients. These abnormalities are believed to be indicative of underlying connectivity deficits and a possible source of the behavioral and cognitive deficits. To evaluate these long-range connectivity and behavioral issues in a cell-autonomous manner, MEK1 loss- and gain-of-function (LoF and GoF) mutations were induced solely in the cortical glutamatergic neurons using a Nex:Cre mouse model. Layer autonomous effects of the cortex were also tested in the GoF mouse using a layer 5 specific Rbp4:Cre mouse. Immunohistochemical analysis showed that activated ERK1/2 (P-ERK1/2) was expressed in high levels in the axonal compartments and reduced levels in the soma when compared to control mice. Axonal tract tracing using a lipophilic dye and an adeno-associated viral (AAV) tract tracing vector, identified significant corticospinal tract (CST) elongation deficits in the LoF and GoF Nex:Cre mouse and in the GoF Rbp4:Cre mouse. AAV tract tracing was further used to identify significant deficits in axonal innervation of the contralateral cortex, the dorsal striatum, and the hind brain of the Nex:Cre GoF mouse and the contralateral cortex and dorsal striatum of the Rbp4:Cre mouse. Behavioral testing of the Nex:Cre GoF mouse indicated deficits in motor learning acquisition while the Rbp4:Cre GoF mouse showed no failure to acquire motor skills as tested. Analysis of the expression levels of the immediate early gene ARC in Nex:Cre and Rbp4:Cre mice showed a specific reduction in a cell- and layer-autonomous manner. These findings suggest that hyperactivation of the RAS/MAPK pathway in cortical glutamatergic neurons, induces changes to the expression patterns of P-ERK1/2, disrupts axonal elongation and innervation patterns, and disrupts motor learning abilities.
ContributorsBjorklund, George Reed (Author) / Newbern, Jason M (Thesis advisor) / Neisewander, Janet (Committee member) / Smith, Brian (Committee member) / Orchinik, Miles (Committee member) / Mangone, Marco (Committee member) / Arizona State University (Publisher)
Created2018
Description
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
A series of mitochondria targeting probes was synthesized for the purpose of exploring the feasibility of a mitochondria targeting fluorescent sensor. Of the probes, the probe with a two carbon spacer showed the best co-localization from staining with the established MitoTracker Red® FM, indicating a potential development of the probe into mitochondria targeting sensor. However, cytotoxicity was observed for the probe with a six carbon spacer. Three additional mitochondria targeting fluorescent probes of longer spacer groups were synthesized, but the cytotoxicity was not observed to be as high as that of the probe with a two carbon spacer. The cytotoxicity was characterized to be that of caspase dependent cell death. To screen for a possible effect on apoptosis due to the mitochondrial probe, three fluorescent fusion proteins binding the anti-apoptotic proteins were designed and expressed. Each purified fusion protein was then incubated with the cytotoxic mitochondrial probe, and the mixture was isolated by running an affinity column. The fluorescence analysis of eluted fractions showed preliminary data of possible interaction between the protein and the mitochondrial probe.
ContributorsLee, Fred (Author) / Meldrum, Deirdre R. (Thesis director) / Tian, Yanqing (Committee member) / Zhang, Liqiang (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-12
Description
An in-depth analysis of Homeview Realty and Homeview Financial was conducted. A marketing plan for both companies was prepared for this project. Homeview Realty and Homeview Financial are in the midst of dynamic industries. The landscape of doing business in the real estate and mortgage industries are constantly changing and evolving. Thus, it is vital for Homeview Realty and Homeview Financial to constantly be knowledgeable in these fields. With this dynamic aspect, the landscape for marketing has also changed; it became digital in nature. Thus, it is important to analyze Homeview Realty and Homeview Financial currently and create a live marketing plan that can be updated when needed. With a marketing plan in hand, Homeview will be able understand its business model, mission, goals, and objectives and in turn be able to create marketing campaigns compatible with the companies objectives and strategic directions.
ContributorsCrowley, Rachel Victoria (Author) / Ostrom, Lonnie (Thesis director) / Montoya, Detra (Committee member) / Mirshak, Paul (Committee member) / Barrett, The Honors College (Contributor) / Department of Marketing (Contributor) / Department of Supply Chain Management (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Department of Finance (Contributor) / School of Accountancy (Contributor)
Created2014-05
Description
Repeating tiles made of DNA were used to try to form an indefinitely large structure. Both the tiles and structure were 2D. Two different patterns were tested, one corrugated and one not. Corrugation means that the tiles alternated between facing up and facing down, canceling out any curvature to the tile and creating a slightly corrugated but largely 2D pattern. Annealing methods were also experimented with. Annealing the structure in two, separate steps as opposed to one was tested. Another experiment was comparing cyclic versus linear annealing. A linear decrease in temperatures defines the linear annealing, and a cyclic method involved a linear drop to a certain temperature, followed by a slight increase in temperature and cooling back down again. This cycle is done several times before it continues linear cool down. It was seen that both corrugated and non-corrugated structures could be made. In both cases tiles that make up a larger section of the overall pattern were more successful. This is especially important for the non-corrugated pattern. Linear and 2step annealing methods seem to yield the best results.
ContributorsHunt, Ashley Elizabeth (Author) / Yan, Liu (Thesis director) / Yan, Hao (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
Lung cancer is the leading cause of cancer-related deaths in the US. Low-dose computed tomography (LDCT) scans are speculated to reduce lung cancer mortality. However LDCT scans impose multiple risks including false-negative results, false- positive results, overdiagnosis, and cancer due to repeated exposure to radiation. Immunosignaturing is a new method proposed to screen and detect lung cancer, eliminating the risks associated with LDCT scans. Known and blinded primary blood sera from participants with lung cancer and no cancer were run on peptide microarrays and analyzed. Immunosignatures for each known sample collectively indicated 120 peptides unique to lung cancer and non-cancer participants. These 120 peptides were used to determine the status of the blinded samples. Verification of the results from Vanderbilt is pending.
ContributorsNguyen, Geneva Trieu (Author) / Woodbury, Neal (Thesis director) / Zhao, Zhan-Gong (Committee member) / Stafford, Phillip (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Department of Psychology (Contributor)
Created2015-05