Matching Items (147)
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
The objective of this thesis was to determine whether Zika Virus (ZIKV) can be effectively inactivated by Selective Photonic Disinfection (SEPHODIS) and determine whether key proteins involved in the infection process are preserved, making SEPHODIS a possible source for vaccine development. As of January 2018, there have been 3,720 confirmed cases of Congenital Zika Syndrome in infants, making a Zika Vaccine a high priority (Mitchell, 2018). SEPHODIS is a process that involves prolonged exposure of an object to a pulsing laser which can render it ineffective. Initially, ZIKV was subjected to laser inactivation for 6 hours, then a plaque assay was performed on both laser-treated and control samples. ZIKV was inactivated two-fold? after laser treatment, when compared with control, as indicated by the plaque assay results. Additionally, both samples were submitted to ELISA to evaluate antigenicity with a panel of monoclonal and human sera. As a second control, virus inactivated by formaldehyde (2%) was used. ELISA results showed that antigenicity of some proteins were preserved while others were probably disturbed. However, ELISA results show that ZIKV envelope protein (E-protein), the protein responsible for viral entry into cells, was effectively preserved after laser-treatment, implying that if laser parameters were tweaked to obtain more complete inactivation, then SEPHODIS may be an appropriate source for the development of a vaccine.
ContributorsViafora, Ataiyo Blue (Author) / Johnston, Stephen (Thesis director) / Tsen, Kong-Thon (Committee member) / School of Life Sciences (Contributor) / School of Sustainability (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Cancer is one of the leading causes of death globally according to the World Health Organization. Although improved treatments and early diagnoses have reduced cancer related mortalities, metastatic disease remains a major clinical challenge. The local tumor microenvironment plays a significant role in cancer metastasis, where tumor cells respond and adapt to a plethora of biochemical and biophysical signals from stromal cells and extracellular matrix (ECM) proteins. Due to these complexities, there is a critical need to understand molecular mechanisms underlying cancer metastasis to facilitate the discovery of more effective therapies. In the past few years, the integration of advanced biomaterials and microengineering approaches has initiated the development of innovative platform technologies for cancer research. These technologies enable the creation of biomimetic in vitro models with physiologically relevant (i.e. in vivo-like) characteristics to conduct studies ranging from fundamental cancer biology to high-throughput drug screening. In this review article, we discuss the biological significance of each step of the metastatic cascade and provide a broad overview on recent progress to recapitulate these stages using advanced biomaterials and microengineered technologies. In each section, we will highlight the advantages and shortcomings of each approach and provide our perspectives on future directions.
ContributorsPeela, Nitish (Author) / Nikkhah, Mehdi (Thesis director) / LaBaer, Joshua (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Glioblastoma is the most aggressive and lethal brain tumor, due to its resistance to current conventional therapy. The resistance to chemo- and radiotherapy has been attributed to a special population of cells known as glioma stem cells. Previous literature has shown the importance of a Central Nervous System-restricted transcription factor OLIG2 in maintaining the tumor-propagating potential of these glioma stem cells. OLIG2's function was further elucidated, with its pro-mitogenic function due to its ability to negatively regulate the p53 pathway by suppressing the acetylation of the p53 protein's C terminal domain. Past work in our lab has confirmed that one of OLIG2's partner proteins is Histone Deacetylase 1 (HDAC1). In vitro experiments have also shown that targeting HDAC1 using hairpin RNA in glioma stem cells negatively impacts proliferation. In a survival study using a murine glioma model, targeting Hdac1 using hairpin RNA is shown to reduce tumor burden and increase survival. In this paper, we demonstrate that silencing Hdac1 expression reduces proliferation, increases cell death, likely a result of increased acetylation of p53. Olig2 expression levels seem to be unaffected in GSCs, demonstrating that the Hdac1 protein ablation is indeed lethal to GSCs. This work builds upon previously collected results, confirming that Hdac1 is a potential surrogate target for Olig2's pro-mitotic function in regulating the p53 pathway.
ContributorsLoo, Vincent You Wei (Author) / LaBaer, Joshua (Thesis director) / Mehta, Shwetal (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
CREB3L1 has been previously shown to auto-acetylate itself when prepared from HeLa cell based in vitro protein expression lysates. To circumvent the concerns of the contamination of co-purified human proteins from HeLa lysates, the protein was purified through insect cell transfection in vitro. The objective of this study was to assay the auto-acetylation activity of CREB3L1 prepared from insect cells using the baculovirus expression vector system (BEVS). To this end, His-tagged CREB3L1 was affinity purified from Hi5 cells using an IMAC column and used for acetylation assay. Samples were taken different time points and auto-acetylation was by western using antibodies specific to acetylated lysines. Auto-acetylation activity was observed after overnight incubation. Future experiments will focus on the improvement of purification yield and the identification of the substrates and interacting proteins of CREB3L1 to better understand the biological functions of this novel acetyltransferase.
ContributorsSchwab, Anna (Author) / LaBaer, Joshua (Thesis director) / Qiu, Ji (Committee member) / Barrett, The Honors College (Contributor)
Created2017-05
Description
PD-L1 blockade has shown recent success in cancer therapy and cancer vaccine regimens. One approach for anti-PD-L1 antibodies has been their application as adjuvants for cancer vaccines. Given the disadvantages of such antibodies, including long half-life and adverse events related to their use, a novel strategy using synbodies in place of antibodies can be tested. Synbodies offer a variety of advantages, including shorter half-life, smaller size, and cheaper cost. Peptides that could bind PD-L1 were identified via peptide arrays and used to construct synbodies. These synbodies were tested with inhibition ELISA assays, SPR, and pull down assays. Additional flow cytometry analysis was done to determine the binding specificity of the synbodies to PD-L1 and the ability of those synbodies to inhibit the PD-L1/PD-1 interaction. Although analysis of permeabilized cells expressing PD-L1 indicated that the synbodies could successfully bind PD-L1, those results were not replicated in non-permeabilized cells. Further assays suggested that the binding of the synbodies was non-specific. Other tests were done to see if the synbodies could inhibit the PD-1/PD-L1 interaction. This assay did not yield any conclusive results and further experimentation is needed to determine the efficacy of the synbodies in inhibiting this interaction.
ContributorsMujahed, Tala (Author) / Johnston, Stephen (Thesis director) / Blattman, Joseph (Committee member) / Diehnelt, Chris (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Finding life beyond Earth could change our understanding of life and habitability. The best place to look for life beyond Earth is Jupiter's moon, Europa. It has been estimated Europa may have a liquid, salt-water subsurface with 2 to 3 times the volume of all Earth's oceans. Knowing that all life requires water, it is in our best interest to explore Europa. This thesis explored the plausibility of life on Europa in four of its environments: on the surface, under the ice shell, in the liquid subsurface, and at the bottom of the liquid subsurface. Each of these environments were defined from science literature and compared to known Earth analogs. Europa's surface is not likely to support life, as there is not liquid water present. There is also extremely high radiation bombardment and extremely low surface temperatures that are estimated to be well out of the range for supporting life. It is more plausible that life could be under Europa's ice shell than on the surface. Under the surface, radiation exposure dramatically reduces. Researchers have found organisms on Earth that can live in similar environments as Europa's ice as well. These organisms require some interaction with liquid water though. Uncertainties about Europa's ice shell thickness and radiation load per depth it experiences, as well as there being limited research on organisms in ice environments, hinder us from definitively assessing the plausibility of life under the surface. The best environment on Europa to look for life on Europa is the subsurface. There remain a lot of uncertainties about the subsurface, however, that make it difficult to assess the plausibility of finding life. These uncertainties include its depth, water activity, salinity, temperature, pressure, and structure. This subsurface may be suitable for life, but until we can further understand the environment of the subsurface, we cannot make definite conclusions. As for assessing the plausibility of life at the bottom of Europa's subsurface, there is not much we know about this environment either. It has been suggested there may be hydrothermal vents, but no evidence has either supported or rejected this idea. Without a clear understanding of the environment at the bottom of the subsurface, the plausibility of life here cannot be definitively answered. It is apparent we need to further study Europa. In particular, we need to focus on understanding the subsurface. When the subsurface is better defined, we can better assess the plausibility of life being present. Fortunately, both NASA and the ESA are currently planning missions to Europa that are scheduled to launch in the 2020s.
ContributorsHoward, Cheyenne Whiffen (Author) / Farmer, Jack (Thesis director) / Shock, Everett (Committee member) / School of Earth and Space Exploration (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
The devastating 2014 Ebola virus outbreak in Western Africa demonstrated the lack of therapeutic approaches available for the virus. Although monoclonal antibodies (mAb) and other molecules have been developed that bind the virus, no therapeutic has shown the efficacy needed for FDA approval. Here, a library of 50 peptide based ligands that bind the glycoprotein of the Zaire Ebola virus (GP) were developed. Using whole virus screening of vesicular stomatitis virus pseudotyped with GP, low affinity peptides were identified for ligand construction. In depth analysis showed that two of the peptide based molecules bound the Zaire GP with <100 nM KD. One of these two ligands was blocked by a known neutralizing mAb, 2G4, and showed cross-reactivity to the Sudan GP. This work presents ligands with promise for therapeutic applications across multiple variants of the Ebola virus.
ContributorsRabinowitz, Joshua Avraam (Author) / Diehnelt, Chris (Thesis director) / Johnston, Stephen (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Both technological and scientific fields continue to revolutionize in a similar fashion; however, a major difference is that high-tech corporations have found models to continue progressions while still keeping product costs low. The main objective was to identify which, if any, components of certain technological models could be used with the vaccine and pharmaceutical markets to significantly lower their costs. Smartphones and computers were the two main items investigated while the two main items from the scientific standpoint were vaccines and pharmaceuticals. One concept had the ability to conceivably decrease the costs of vaccines and drugs and that was "market competition". If the United States were able to allow competition within the vaccine and drug companies, it would allow for the product prices to be best affected. It would only take a few small companies to generate generic versions of the drugs and decrease the prices. It would force the larger competition to most likely decrease their prices. Furthermore, the PC companies use a cumulative density function (CDF) to effectively divide their price setting in each product cycle. It was predicted that if this CDF model were applied to the vaccine and drug markets, the prices would no longer have to be extreme. The corporations would be able to set the highest price for the wealthiest consumers and then slowly begin to decrease the costs for the middle and lower class. Unfortunately, the problem within the vaccine and pharmaceutical markets was not the lack of innovation or business models. The problem lied with their liberty to choose product costs due to poor U.S. government regulations.
ContributorsCalderon, Gerardo (Author) / Johnston, Stephen (Thesis director) / Diehnelt, Chris (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Almost every form of cancer deregulates the expression and activity of anabolic glycosyltransferase (GT) enzymes, which incorporate particular monosaccharides in a donor acceptor as well as linkage- and anomer-specific manner to assemble complex and diverse glycans that significantly affect numerous cellular events, including tumorigenesis and metastasis. Because glycosylation is not template-driven, GT deregulation yields heterogeneous arrays of aberrant intact glycan products, some in undetectable quantities in clinical bio-fluids (e.g., blood plasma). Numerous glycan features (e.g., 6 sialylation, β-1,6-branching, and core fucosylation) stem from approximately 25 glycan “nodes:” unique linkage specific monosaccharides at particular glycan branch points that collectively confer distinguishing features upon glycan products. For each node, changes in normalized abundance (Figure 1) may serve as nearly 1:1 surrogate measure of activity for culpable GTs and may correlate with particular stages of carcinogenesis. Complementary to traditional top down glycomics, the novel bottom-up technique applied herein condenses each glycan node and feature into a single analytical signal, quantified by two GC-MS instruments: GCT (time-of-flight analyzer) and GCMSD (transmission quadrupole analyzers). Bottom-up analysis of stage 3 and 4 breast cancer cases revealed better overall precision for GCMSD yet comparable clinical performance of both GC MS instruments and identified two downregulated glycan nodes as excellent breast cancer biomarker candidates: t-Gal and 4,6-GlcNAc (ROC AUC ≈ 0.80, p < 0.05). Resulting from the activity of multiple GTs, t-Gal had the highest ROC AUC (0.88) and lowest ROC p‑value (0.001) among all analyzed nodes. Representing core-fucosylation, glycan node 4,6-GlcNAc is a nearly 1:1 molecular surrogate for the activity of α-(1,6)-fucosyltransferase—a potential target for cancer therapy. To validate these results, future projects can analyze larger sample sets, find correlations between breast cancer stage and changes in t-Gal and 4,6-GlcNAc levels, gauge the specificity of these nodes for breast cancer and their potential role in other cancer types, and develop clinical tests for reliable breast cancer diagnosis and treatment monitoring based on t-Gal and 4,6-GlcNAc.
ContributorsZaare, Sahba (Author) / Borges, Chad (Thesis director) / LaBaer, Joshua (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05