Matching Items (121)
Description
The purpose of this project was to identify proteins associated with the migration and invasion of non-transformed MCF10A mammary epithelial cells with ectopically expressed missense mutations in p53. Because of the prevalence of TP53 missense mutations in basal-like and triple-negative breast cancer tumors, understanding the effect of TP53 mutations on the phenotypic expression of human mammary epithelial cells may offer new therapeutic targets for those currently lacking in treatment options. As such, MCF10A mammary epithelial cells ectopically overexpressing structural mutations (G245S, H179R, R175H, Y163C, Y220C, and Y234C) and DNA-binding mutations (R248Q, R248W, R273C, and R273H) in the DNA-binding domain were selected for use in this project. Overexpression of p53 in the mutant cell lines was confirmed by western blot and q-PCR analysis targeting the V5 epitope tag present in the pLenti4 vector used to transduce TP53 into the mutant cell lines. Characterization of the invasion and migration phenotypes resulting from the overexpression of p53 in the mutant cell lines was achieved using transwell invasion and migration assays with Boyden chambers. Statistical analysis showed that three cell lines—DNA-contact mutants R248W and R273C and structural mutant Y220C—were consistently more migratory and invasive and demonstrated a relationship between the migration and invasion properties of the mutant cell lines. Two families of proteins were then explored: those involved in the Epithelial-Mesenchymal Transition (EMT) and matrix metalloproteinases (MMPs). Results of q-PCR and immunofluorescence analysis of epithelial marker E-cadherin and mesenchymal proteins Slug and Vimentin did not show a clear relationship between mRNA and protein expression levels with the migration and invasiveness phenotypes observed in the transwell studies. Results of western blotting, q-PCR, and zymography of MMP-2 and MMP-9 also did not show any consistent results indicating a definite relationship between MMPs and the overall invasiveness of the cells. Finally, two drugs were tested as possible treatments inhibiting invasiveness: ebselen and SBI-183. These drugs were tested on only the most invasive of the MCF10A p53 mutant cell lines (R248W, R273C, and Y220C). Results of invasion assay following 30 μM treatment with ebselen and SBI-183 showed that ebselen does not inhibit invasiveness; SBI-183, however, did inhibit invasiveness in all three cell lines tested. As such, SBI-183 will be an important compound to study in the future as a treatment that could potentially serve to benefit triple-negative or basal-like breast cancer patients who currently lack therapeutic treatment options.
ContributorsZhang, Kathie Q (Author) / LaBaer, Joshua (Thesis director) / Anderson, Karen (Committee member) / Gonzalez, Laura (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
In oxygenic photosynthesis, Photosystem I (PSI) and Photosystem II (PSII) are two transmembrane protein complexes that catalyze the main step of energy conversion; the light induced charge separation that drives an electron transfer reaction across the thylakoid membrane. Current knowledge of the structure of PSI and PSII is based on three structures: PSI and PSII from the thermophilic cyanobacterium Thermosynechococcus elonagatus and the PSI/light harvesting complex I (PSI-LHCI) of the plant, Pisum sativum. To improve the knowledge of these important membrane protein complexes from a wider spectrum of photosynthetic organisms, photosynthetic apparatus of the thermo-acidophilic red alga, Galdieria sulphuraria and the green alga, Chlamydomonas reinhardtii were studied. Galdieria sulphuraria grows in extreme habitats such as hot sulfur springs with pH values from 0 to 4 and temperatures up to 56°C. In this study, both membrane protein complexes, PSI and PSII were isolated from this organism and characterized. Ultra-fast fluorescence spectroscopy and electron microscopy studies of PSI-LHCI supercomplexes illustrate how this organism has adapted to low light environmental conditions by tightly coupling PSI and LHC, which have not been observed in any organism so far. This result highlights the importance of structure-function relationships in different ecosystems. Galdieria sulphuraria PSII was used as a model protein to show the amenability of integral membrane proteins to top-down mass spectrometry. G.sulphuraria PSII has been characterized with unprecedented detail with identification of post translational modification of all the PSII subunits. This study is a technology advancement paving the way for the usage of top-down mass spectrometry for characterization of other large integral membrane proteins. The green alga, Chlamydomonas reinhardtii is widely used as a model for eukaryotic photosynthesis and results from this organism can be extrapolated to other eukaryotes, especially agricultural crops. Structural and functional studies on the PSI-LHCI complex of C.reinhardtii grown under high salt conditions were studied using ultra-fast fluorescence spectroscopy, circular dichroism and MALDI-TOF. Results revealed that pigment-pigment interactions in light harvesting complexes are disrupted and the acceptor side (ferredoxin docking side) is damaged under high salt conditions.
ContributorsThangaraj, Balakumar (Author) / Fromme, Petra (Thesis advisor) / Shock, Everett (Committee member) / Chen, Julian (Committee member) / Arizona State University (Publisher)
Created2010
Description
Tempe Town Lake is the site of fifteen years’ worth of chemical data collection by ASU researchers. In 2018 the dataSONDE, an instrument capable of measuring different water quality parameters every thirty minutes for a month at a time was installed in the lake. The SONDE has the potential to completely reduce the need for sampling by hand. Before the SONDE becomes the sole means of gathering data, it is important to verify its accuracy. In this study, the measurements gathered by the SONDE (pH, dissolved oxygen, temperature, conductivity and colored dissolved organic matter) were compared to measurements gathered using the verified methods from the past fifteen years.
ContributorsSauer, Elinor Rayne (Author) / Hartnett, Hilairy (Thesis director) / Glaser, Donald (Committee member) / Shock, Everett (Committee member) / Historical, Philosophical & Religious Studies (Contributor) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / School of Sustainability (Contributor) / Barrett, The Honors College (Contributor)
Created2020-12
Description
The membrane proximal region (MPR, residues 649–683) and transmembrane domain (TMD, residues 684–705) of the gp41 subunit of HIV-1’s envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662–683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649–705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM).
Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.
ContributorsGong, Zhen (Author) / Martin Garcia, Jose Manuel (Author) / Daskalova, Sasha (Author) / Craciunescu, Felicia (Author) / Song, Lusheng (Author) / Dorner, Katerina (Author) / Hansen, Debra (Author) / Yang, Jay-How (Author) / LaBaer, Joshua (Author) / Hogue, Brenda (Author) / Mor, Tsafrir (Author) / Fromme, Petra (Author) / Department of Chemistry and Biochemistry (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Infectious Diseases and Vaccinology (Contributor) / Innovations in Medicine (Contributor) / Personalized Diagnostics (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor)
Created2015-08-21
Description
Dissolved organic matter (DOM) can have numerous effects on the water chemistry and the biological life within an aquatic system with its wide variety of chemical structures and properties. The composition of the dissolved carbon can be estimated by utilizing the fluorescent properties of some DOM such as aromatic amino acids and humic material. This experiment was used to observe how organic matter could influence hydrothermal systems, such as Sylvan Springs in Yellowstone National Park, USA. Using optical density at 600 nm (OD 600), excitation-emission matrix spectra (EEMS), and Illumina sequencing methods (16S rRNA gene sequencing), changes in dissolved organic matter (DOM) were observed based on long term incubation at 84ºC and microbial influence. Four media conditions were tested over a two-month duration to assess these changes: inoculated pine needle media, uninoculated pine needle media, inoculated yeast extract media, and uninoculated yeast extract media. The inoculated samples contained microbes from a fluid and sediment sample of Sylvan Spring collected July 23, 2018. Absorbance indicated that media containing pine needle broth poorly support life, whereas media containing yeast extract revealed a positive increase in growth. Excitation-Emission Matrix Spectra of the all media conditions indicated changes in DOM composition throughout the trial. There were limited differences between the inoculated and uninoculated samples suggesting that the DOM composition change in this study was dominated by the two-month incubation at 84ºC more than biotic processes. Sequencing performed on a sediment sample collected from Sylvan Spring indicated five main order of prokaryotic phyla: Aquificales, Desulfurococcales, Thermoproteales, Thermodesulfobacteriales, and Crenarchaeota. These organisms are not regarded as heterotrophic microbes, so the lack of significant biotic changes in DOM could be a result of these microorganisms not being able to utilize these enrichments as their main metabolic energy supply.
ContributorsKnott, Nicholas Joseph (Author) / Shock, Everett (Thesis director) / Hartnett, Hilairy (Committee member) / Till, Christy (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The purpose of this thesis creative project was to create an educational video to present research findings on the increasingly important issue of human biospecimen preanalytic variables. When a human biospecimen, such as blood, urine, or tissue, is removed from the body, it is subjected to a plethora of variables that are not recorded or regulated in a vast majority of cases. Frequently, these samples arrive at the research or pathology lab with an unknown history, then undergo analysis for translational research purposes, or to guide clinical management decisions. Thus, compromised specimen quality caused by preanalytic variables has substantial, and potentially devastating, downstream effects. To identify the preanalytic variables with the greatest impact on blood and tissue specimen quality, 45 articles were gathered using PubMed and Google Scholar databases and cited. Based on the articles, the top five variables with the most detrimental effects were identified for both blood and tissue samples. Multiple sets of parameters ensuring specimen fitness were compared for each of the five variables for each specimen type. Then, specific parameters guaranteeing the fitness of the greatest number of analytes were verified. To present the research findings in greater detail, a paper was written that focused on identifying the top variables and key parameters to ensure analyte fitness. To present the overall issue in an easy-to-digest format, a storyboard and script were created as a guideline for a final video project. Ultimately, two alternate versions of the video were created to pertain to the audience of choice (one version for patients, one version for professionals). It is the hope that these videos will be used as educational tools to continue efforts to standardize and enforce human biospecimen preanalytic variable parameters. This is a necessary step to improve the accuracy of our biomedical research data and the healthcare of patients worldwide.
ContributorsAzcarate, Heather (Author) / Compton, Carolyn (Thesis director) / LaBaer, Joshua (Committee member) / Borges, Chad (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor)
Created2018-12
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
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