Matching Items (118)
Description
DNA methylation, a subset of epigenetics, has been found to be a significant marker associated with variations in gene expression and activity across the entire human genome. As of now, however, there is little to no information about how DNA methylation varies between different tissues inside a singular person's body. By using research data from a preliminary study of lean and obese clinical subjects, this study attempts to put together a profile of the differences in DNA methylation that can be observed between two particular body tissues from this subject group: blood and skeletal muscle. This study allows us to start describing the changes that occur at the epigenetic level that influence how differently these two tissues operate, along with seeing how these tissues change between individuals of different weight classes, especially in the context of the development of symptoms of Type 2 Diabetes.
ContributorsRappazzo, Micah Gabriel (Author) / Coletta, Dawn (Thesis director) / Katsanos, Christos (Committee member) / Dinu, Valentin (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor) / Department of Psychology (Contributor)
Created2013-12
Description
Protein folding is essential in all cells, and misfolded proteins cause many diseases. In the Gram-negative bacterium Escherichia coli, protein folding must be carefully controlled during envelope biogenesis to maintain an effective permeability barrier between the cell and its environment. This study explores the relationship between envelope biogenesis and cell stress, and the return to homeostasis during envelope stress. A major player in envelope biogenesis and stress response is the periplasmic protease DegP. Work presented here explores the growth phenotypes of cells lacking degP, including temperature sensitivity and lowered cell viability. Intriguingly, these cells also accumulate novel cytosolic proteins in their envelope not present in wild-type. Association of novel proteins was found to be growth time- and temperature-dependent, and was reversible, suggesting a dynamic nature of the envelope stress response. Two-dimensional gel electrophoresis of envelopes followed by mass spectrometry identified numerous cytoplasmic proteins, including the elongation factor/chaperone TufA, illuminating a novel cytoplasmic response to envelope stress. A suppressor of temperature sensitivity was characterized which corrects the defect caused by the lack of degP. Through random Tn10 insertion analysis, aribitrarily-primed polymerase chain reaction and three-factor cross, the suppressor was identified as a novel duplication-truncation of rpoE, here called rpoE'. rpoE' serves to subtly increase RpoE levels in the cell, resulting in a slight elevation of the SigmaE stress response. It does so without significantly affecting steady-state levels of outer membrane proteins, but rather by increasing proteolysis in the envelope independently of DegP. A multicopy suppressor of temperature sensitivity in strains lacking degP and expressing mutant OmpC proteins, yfgC, was characterized. Bioinformatics suggests that YfgC is a metalloprotease, and mutation of conserved domains resulted in mislocalization of the protein. yfgC-null mutants displayed additive antibiotic sensitivity and growth defects when combined with null mutation in another periplasmic chaperone, surA, suggesting that the two act in separate pathways during envelope biogenesis. Overexpression of YfgC6his altered steady-state levels of mutant OmpC in the envelope, showing a direct relationship between it and a major constituent of the envelope. Curiously, purified YfgC6his showed an increased propensity for crosslinking in mutant, but not in a wild-type, OmpC background.
ContributorsLeiser, Owen Paul (Author) / Misra, Rajeev (Thesis advisor) / Jacobs, Bertram (Committee member) / Chang, Yung (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2010
Description
Coronaviruses are medically important viruses that cause respiratory and enteric infections in humans and animals. The recent emergence through interspecies transmission of severe acute respiratory syndrome coronavirus (SARS-CoV) strongly supports the need for development of vaccines and antiviral reagents. Understanding the molecular details of virus assembly is an attractive target for development of such therapeutics. Coronavirus membrane (M) proteins constitute the bulk of the viral envelope and play key roles in assembly, through M-M, M-spike (S) and M-nucleocapsid (N) interactions. M proteins have three transmembrane domains, flanked by a short amino-terminal domain and a long carboxy-terminal tail located outside and inside the virions, respectively. Two domains are apparent in the long tail - a conserved region (CD) at the amino end and a hydrophilic, charged carboxy-terminus (HD). We hypothesized that both domains play functionally important roles during assembly. A series of changes were introduced in the domains and the functional impacts were studied in the context of the virus and during virus-like particle (VLP) assembly. Positive charges in the CD gave rise to viruses with neutral residue replacements that exhibited a wild-type phenotype. Expression of the mutant proteins showed that neutral, but not positive, charges formed VLPs and coexpression with N increased output. Alanine substitutions resulted in viruses with crippled phenotypes and proteins that failed to assemble VLPs or to be rescued into the envelope. These viruses had partially compensating changes in M. Changes in the HD identified a cluster of three key positive charges. Viruses could not be recovered with negatively charged amino acid substitutions at two of the positions. While viruses were recovered with a negative charge substitution at one of the positions, these exhibited a severely crippled phenotype. Crippled mutants displayed a reduction in infectivity. Results overall provide new insight into the importance of the M tail in virus assembly. The CD is involved in fundamental M-M interactions required for envelope formation. These interactions appear to be stabilized through interactions with the N protein. Positive charges in the HD also play an important role in assembly of infectious particles.
ContributorsArndt, Ariel L (Author) / Hogue, Brenda G (Thesis advisor) / Jacobs, Bertram (Committee member) / Francisco, Wilson (Committee member) / Ugarova, Tatiana (Committee member) / Arizona State University (Publisher)
Created2010
Description
Host organisms have evolved multiple mechanisms to defend against a viral infection and likewise viruses have evolved multiple methods to subvert the host's anti-viral immune response. Vaccinia virus (VACV) is known to contain numerous proteins involved in blocking the cellular anti-viral immune response. The VACV E3L protein is important for inhibiting the anti-viral immune response and deletions within this gene lead to a severe attenuation. In particular, VACV containing N-terminal truncations in E3L are attenuated in animal models and fail to replicate in murine JC cells. Monkeypox virus (MPXV) F3L protein is a homologue of the VACV E3L protein, however it is predicted to contain a 37 amino acid N-terminal truncation. Despite containing an N-terminal truncation in the E3L homologue, MPXV is able to inhibit the anti-viral immune response similar to wild-type VACV and able to replicate in JC cells. This suggests that MPXV has evolved another mechanism(s) to counteract host defenses and promote replication in JC cells. MPXV produces less dsRNA than VACV during the course of an infection, which may explain why MPXV posses a phenotype similar to VACV, despite containing a truncated E3L homologue. The development of oncolytic viruses as a therapy for cancer has gained interest in recent years. Oncolytic viruses selectively replicate in and destroy cancerous cells and leave normal cells unharmed. Many tumors possess dysregulated anti-viral signaling pathways, since these pathways can also regulate cell growth. Creating a mutation in the N-terminus of the VACV-E3L protein generates an oncolytic VACV that depends on dysregulated anti-viral signaling pathways for replication allowing for direct targeting of the cancerous cells. VACV-E3Ldel54N selectively replicates in numerous cancer cells lines and not in the normal cell lines. Additionally, VACV-E3Ldel54N is safe and effective in causing tumor regression in a xenograph mouse model. Lastly, VACV-E3Ldel54N was capable of spreading from the treated tumors to the untreated tumors in both a xenograph and syngeneic mouse model. These data suggest that VACV-E3Ldel54N could be an effective oncolytic virus for the treatment of cancer.
ContributorsArndt, William D (Author) / Jacobs, Bertram (Thesis advisor) / Curtiss Iii, Roy (Committee member) / Chang, Yung (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2010
Description
Lipid membranes are a key structure for many classes of viruses. Lipid membranes can be analyzed using the fluid mosaic model, which states that the phospholipid membrane has variable amounts of fluidity and key membrane proteins are presented in areas stabilized by cholesterol-enriched platforms called lipid rafts. This project aims to further the understanding of the importance of lipid rafts in measles virus (MV) infection and replication, which has not been extensively studied. In order to do this, an MV-susceptible cell line was treated with an anti-cholesterol compound before and after measles virus infection. I found that pre-infection treatments had a marginal effect upon measles cytopathic effect (syncytia formation) or replication. Twenty-four hours post-infection treatment had a deleterious effect on cell viability, but the replication/assembly of infectious units per cell decreased importantly and in dose-dependent manner. Furthermore, by measuring the susceptibility to neutralization of infectious particles obtained from MBCD treated cells, I determined the importance of lipid microdomain environment on the stability of infectious particles. Increased anti-cholesterol treatment enhanced the susceptibility of MV to neutralization. Future studies are proposed to assess the properties of cholesterol depleted viral infectious units.
ContributorsYkema, Matthew Ryan (Author) / Mor, Tsafrir (Thesis director) / Jacobs, Bertram (Committee member) / Julik, Emily (Committee member) / Barrett, The Honors College (Contributor) / Economics Program in CLAS (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.
ContributorsLi, Xuanxuan (Author) / Chiu, Chun-Ya (Author) / Wang, Hsiang-Ju (Author) / Kassemeyer, Stephan (Author) / Botha, Sabine (Author) / Shoeman, Robert L. (Author) / Lawrence, Robert (Author) / Kupitz, Christopher (Author) / Kirian, Richard (Author) / James, Daniel (Author) / Wang, Dingjie (Author) / Nelson, Garrett (Author) / Messerschmidt, Marc (Author) / Boutet, Sebastien (Author) / Williams, Garth J. (Author) / Hartman, Elisabeth (Author) / Jafarpour, Aliakbar (Author) / Foucar, Lutz M. (Author) / Barty, Anton (Author) / Chapman, Henry (Author) / Liang, Mengning (Author) / Menzel, Andreas (Author) / Wang, Fenglin (Author) / Basu, Shibom (Author) / Fromme, Raimund (Author) / Doak, R. Bruce (Author) / Fromme, Petra (Author) / Weierstall, Uwe (Author) / Huang, Michael H. (Author) / Spence, John (Author) / Schlichting, Ilme (Author) / Hogue, Brenda (Author) / Liu, Haiguang (Author) / ASU Biodesign Center Immunotherapy, Vaccines and Virotherapy (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Department of Physics (Contributor) / School of Life Sciences (Contributor)
Created2017-04-11
Description
Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.
ContributorsDeb, Arpan (Author) / Johnson, William (Author) / Kline, Alexander (Author) / Scott, Boston (Author) / Meador, Lydia (Author) / Srinivas, Dustin (Author) / Martin Garcia, Jose Manuel (Author) / Dorner, Katerina (Author) / Borges, Chad (Author) / Misra, Rajeev (Author) / Hogue, Brenda (Author) / Fromme, Petra (Author) / Mor, Tsafrir (Author) / ASU Biodesign Center Immunotherapy, Vaccines and Virotherapy (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor) / Biodesign Institute (Contributor) / School of Molecular Sciences (Contributor) / Applied Structural Discovery (Contributor) / Personalized Diagnostics (Contributor)
Created2017-02-22
Description
With the advent of precision medicine, oncologists aim to target tumors that do not respond well to conventional treatment. One such therapy is oncolytic virotherapy, a treatment reliant on viral replication for tumor specific killing. Downregulation of the proteins RIP3 kinase, DAI or MLKL can result in a nonfunctional programmed necroptotic cell death pathway, common amongst breast cancer and melanoma. Vaccinia virus (VACV) mutants with a nonfunctional E3 protein are able to selectively replicate in necroptosis deficient cells but not in necroptosis competent cells, making them potential candidates for oncolytic virotherapy. In order to establish the efficacy and selectivity of this treatment, an accurate tumor model is required. Eight established breast adenocarcinomas and two established melanomas were selected as potential candidates, both human and murine. A pan screening method for necroptosis was established utilizing western blot analysis for expression of aforementioned proteins following various induction methods such as IFN α or VACV infection. In addition, live cell imaging after treatment with tumor necrosis factor (TNFα) and the pan-caspase inhibitor zVAD-fmk was used as a method to visualize necroptosis pathway functionality. Based on these results, cell lines will be selected and modified to create a breast cancer model with cells that are syngeneic, differing only in expression of either RIP3. VACV can be tested for tumor volume reduction in these models to ask if RIP3 expression affects efficacy of mutant VACV as an oncolytic virus.
ContributorsKumar, Aradhana (Author) / Jacobs, Bertram (Thesis director) / McFadden, Grant (Committee member) / Borad, Mitesh (Committee member) / School of Life Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Novel biological strategies for cancer therapy have recently been able to generate antitumor effects in the clinic. Of these new advancements, oncolytic virotherapy seems to be a promising strategy through a dual mechanism of oncolysis and immunogenicity of the host to the target cells. Myxoma virus (MYXV) is an oncolytic poxvirus that has a natural tropism for European rabbits, being nonpathogenic in humans and all other known vertebrates. MYXV is able to infect cancer cells which, due to mutations, have defects in many signaling pathways, notably pathways involved in antiviral responses. While MYXV alone elicits lysis of cancer cells, recombinant techniques allow for the implementation of transgenes, which have the potential of ‘arming’ the virus to enhance its potential as an oncolytic virus. The implementation of certain transgenes allow for the promotion of robust anti-tumor immune responses. To investigate the potential of immune-inducing transgenes in MYXV, in vitro experiments were performed with several armed recombinant MYXVs as well as unarmed wild-type and rabbit-attenuated MYXV. As recent studies have shown the ability of MYXV to uniquely target malignant human hematopoietic stem cells, the potential of oncolytic MYXV armed with immune-inducing transgenes was investigated through in vitro killing analysis using human acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines. Furthermore, in vitro experiments were also performed using primary bone marrow (BM) cells obtained from human patients diagnosed with MM. In this study, armed MYXV-infected human AML and MM cells resulted in increased cell death relative to unarmed MYXV-infected cells, suggesting enhanced killing via induced mechanisms of cell death from the immune-inducing transgenes. Furthermore, increased killing of primary BM cells with multiple myeloma was seen in armed MYXV-infected primary cells relative to unarmed MYXV-infected primary cells.
ContributorsMamola, Joseph (Author) / McFadden, Grant (Thesis director) / Jacobs, Bertram (Committee member) / Blattman, Joseph (Committee member) / School of Life Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Brought on by extended survival due to Highly Active Anti-Retroviral Therapy and increased incidence among older adults, the demographic profile of the HIV epidemic has begun to shift towards the aging population. As people living with HIV (PLWH) begin to age and develop multiple comorbidities, their needs are no longer limited to HIV treatment and disease management; they may require aging services similar to those with a negative HIV status. Increased attention has been placed on HIV and aging to assess the unique needs of older PLWH, however, limited research exists on the preparedness of aging services to provide adequate care to this population. This study aims to assess HIV and aging within Maricopa County, where individuals aged 50 years and older account for nearly half the reported HIV/AIDS cases in the county, and 30% of cases in Arizona. Two focus groups – one with older PLWH and another with aging service professionals – were conducted to gather information about existing aging services and the perspectives of older PLWH regarding their growing needs. Older PLWH were found to experience challenges similar to those that have been well-documented in previous studies: most notably, PTSD and other mental health conditions; fear of the future and isolation; HIV status disclosure and stigma; and economics and financial security. An anonymous survey was developed in conjunction with Aunt Rita’s Foundation to evaluate Maricopa County aging services; it was discovered that providers lack experience with HIV and admit deficiencies in their preparation to address the age-related concerns of older PLWH. The results show that the majority of providers were supportive of offering care to older PLWH and expressed interest in improving their preparedness. Future research is necessary to obtain perspectives from additional aging services in Maricopa County and word towards the development of an aging services directory to connect older PLWH to care.
ContributorsLayon, Sarah (Author) / Jacobs, Bertram (Thesis director) / Coon, David (Committee member) / Spencer, Glen (Committee member) / School of Life Sciences (Contributor) / School of Human Evolution & Social Change (Contributor) / School of Art (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05