Matching Items (83)
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
Measles is a contagious, vaccine-preventable disease that continues to be the leading
cause of death in children younger than the age of 5 years. While the introduction of the Measles, Mumps, and Rubella vaccine (MMR) has significantly decreased morbidity and mortality rates worldwide, vaccine coverage is highly variable across global regions. Current diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. Commercially available Diamedix Immunosimplicity® Measles IgG test kit has been shown to have 91.1% sensitivity and 93.8% specificity, with a positive predictive value of 88.7% and a negative predictive value of 90.9% on the basis of a PRN titer of 120. There is an increasing need for rapid screening for measles specific immunity in outbreak settings. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to three individual measles virus (MeV) proteins.
Measles virus (MeV) genes were subcloned into the pJFT7_nGST vector to generate N- terminal GST fusion proteins. Single MeV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured with mouse anti-GST mAb and sheep anti-mouse IgG. Relative light units (RLUs) as luminescence was measured. Antibodies to MeV antigens were measured in 40 serum samples from healthy subjects.
Protein expression of three MeV genes of interest was measured in comparison with vector control and statistical significance was determined using the Student’s t-test (p<0.05). N expressed at the highest level with an average RLU value of 3.01 x 109 (p<0.001) and all proteins were expressed at least 50% greater than vector control (4.56 x 106 RLU). 36/40 serum samples had IgG to N (Ag:GST ratio>1.21), F (Ag:GST ratio>1.92), or H (Ag:GST ratio> 1.23).
These data indicate that the in vitro expression of MeV antigens, N, F, and H, were markedly improved by subcloning into pJFT7_nGST vector to generate N-terminal GST fusion proteins. The expression of single MeV genes N, F and H, are suitable antigens for serologic capture analysis of measles-specific antibodies. These preliminary data can be used to design a more intensive study to explore the possibilities of using these MeV antigens as a diagnostic marker.
cause of death in children younger than the age of 5 years. While the introduction of the Measles, Mumps, and Rubella vaccine (MMR) has significantly decreased morbidity and mortality rates worldwide, vaccine coverage is highly variable across global regions. Current diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. Commercially available Diamedix Immunosimplicity® Measles IgG test kit has been shown to have 91.1% sensitivity and 93.8% specificity, with a positive predictive value of 88.7% and a negative predictive value of 90.9% on the basis of a PRN titer of 120. There is an increasing need for rapid screening for measles specific immunity in outbreak settings. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to three individual measles virus (MeV) proteins.
Measles virus (MeV) genes were subcloned into the pJFT7_nGST vector to generate N- terminal GST fusion proteins. Single MeV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured with mouse anti-GST mAb and sheep anti-mouse IgG. Relative light units (RLUs) as luminescence was measured. Antibodies to MeV antigens were measured in 40 serum samples from healthy subjects.
Protein expression of three MeV genes of interest was measured in comparison with vector control and statistical significance was determined using the Student’s t-test (p<0.05). N expressed at the highest level with an average RLU value of 3.01 x 109 (p<0.001) and all proteins were expressed at least 50% greater than vector control (4.56 x 106 RLU). 36/40 serum samples had IgG to N (Ag:GST ratio>1.21), F (Ag:GST ratio>1.92), or H (Ag:GST ratio> 1.23).
These data indicate that the in vitro expression of MeV antigens, N, F, and H, were markedly improved by subcloning into pJFT7_nGST vector to generate N-terminal GST fusion proteins. The expression of single MeV genes N, F and H, are suitable antigens for serologic capture analysis of measles-specific antibodies. These preliminary data can be used to design a more intensive study to explore the possibilities of using these MeV antigens as a diagnostic marker.
ContributorsMushtaq, Zuena (Author) / Anderson, Karen (Thesis advisor) / Blattman, Joseph (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2016
Description
Osteosarcoma is the most common bone cancer in children and adolescents. Patients with metastatic osteosarcoma are typically refractory to treatment. Numerous lines of evidence suggest that cytotoxic T-lymphocytes (CTL) limit the development of metastatic osteosarcoma. I have investigated the role of Programmed Death Receptor-1 (PD-1) in limiting the efficacy of immune mediated control of metastatic osteosarcoma. I show that human metastatic, but not primary, osteosarcoma tumors express the ligand for PD-1 (PD-L1) and that tumor infiltrating CTL express PD-1, suggesting this pathway may limit CTL control of metastatic osteosarcoma in patients. PD-L1 is also expressed on the K7M2 osteosarcoma tumor cell line that establishes metastases in mice, and PD-1 is expressed on tumor infiltrating CTL during disease progression. Blockade of PD-1/PD-L1 interactions dramatically improves the function of osteosarcoma-reactive CTL in vitro and in vivo, and results in decreased tumor burden and increased survival in the K7M2 mouse model of metastatic osteosarcoma. My results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma should be pursued as a therapeutic strategy. However, PD-1/PD-L1 blockade treated mice still succumb to disease due to selection of PD-L1 mAb resistant tumor cells via up-regulation of other co-inhibitory T cell receptors. Combinational α-CTLA-4 and α-PD-L1 blockade treated mice were able to completely eradicate metastatic osteosarcoma, and generate immunity to disease. These results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma, although improves survival, may lead to tumor resistance, requiring combinational immunotherapies to combat and eradicate disease.
ContributorsLussier, Danielle (Author) / Blattman, Joseph N. (Thesis advisor) / Anderson, Karen (Committee member) / Goldstein, Elliott (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2015
Description
The majority of non-small cell lung cancer (NSCLC) patients (70%) are diagnosed with adenocarcinoma versus other histological subtypes. These patients often present with advanced, metastatic disease and frequently relapse after treatment. The tumor suppressor, Liver Kinase B1, is frequently inactivated in adenocarcinomas and loss of function is associated with a highly aggressive, metastatic tumor (1). Identification of the mechanisms deregulated with LKB1 inactivation could yield targeted therapeutic options for adenocarcinoma patients. Re-purposing the immune system to support tumor growth and aid in metastasis has been shown to be a feature in cancer progression (2). Tumor associated macrophages (TAMs) differentiate from monocytes, which are recruited to the tumor microenvironment via secretion of chemotaxic factors by cancer cells. We find that NSCLC cells deficient in LKB1 display increased secretion of C-C motif ligand 2 (CCL2), a chemokine involved in monocyte recruitment. To elucidate the molecular pathway regulating CCL2 up-regulation, we investigated inhibitors of substrates downstream of LKB1 signaling in A549, H23, H2030 and H838 cell lines. Noticeably, BAY-11-7082 (NF-κB inhibitor) reduced CCL2 secretion by an average 92%. We further demonstrate that a CCR2 antagonist and neutralizing CCL2 antibody substantially reduce monocyte migration to NSCLC (H23) cell line conditioned media. Using an in vivo model of NSCLC, we find that LKB1 deleted tumors demonstrate a discernible increase in CCL2 levels compared to normal lung. Moreover, tumors display an increase in the M2:M1 macrophage ratio and increase in tumor associated neutrophil (TAN) infiltrate compared to normal lung. This M2 shift was significantly reduced in mice treated with anti-CCL2 or a CCR2 antagonist and the TAN infiltrate was significantly reduced with the CCR2 antagonist. These data suggest that deregulation of the CCL2/CCR2 signaling axis could play a role in cancer progression in LKB1 deficient tumors.
ContributorsFriel, Jacqueline (Author) / Inge, Landon (Thesis advisor) / Lake, Douglas (Thesis advisor) / Blattman, Joseph (Committee member) / Arizona State University (Publisher)
Created2015
Description
Skeletal muscle injury may occur from repetitive short bursts of biomechanical strain that impair muscle function. Alternatively, variations of biomechanical strain such as those held for long-duration are used by clinicians to repair muscle and restore its function. Fibroblasts embedded within the unifying connective tissue of skeletal muscle experience these multiple and diverse mechanical stimuli and respond by secreting cytokines. Cytokines direct all stages of muscle regeneration including myoblasts differentiation, fusion to form myotubes, and myotube functionality. To examine how fibroblasts respond to variations in mechanical strain that may affect juxtapose muscle, a myofascial junction was bioengineered that examined the interaction between the two cell types. Fibroblasts were experimentally shown to increase myoblast differentiation, and fibroblast biomechanical strain mediated the extent to which differentiation occurred. Intereleukin-6 is a strain-regulated cytokine secreted by fibroblasts was determined to be necessary for fibroblast-mediated myoblast differentiation. Myotubes differentiated in the presence of strained fibroblasts express greater number of acetylcholine receptors, greater acetylcholine receptor sizes, and modified to be more or less sensitive to acetylcholine-induced contraction. This study provides direct evidence that strained and non-strained fibroblasts can serve as a vehicle to modify myoblast differentiation and myotube functionality. Further understanding the mechanisms regulating these processes may lead to clinical interventions that include strain-activated cellular therapies and bioengineered cell engraftment for mediating the regeneration and function of muscle in vivo.
ContributorsHicks, Michael (Author) / Standley, Paul R (Thesis advisor) / Rawls, Jeffrey (Committee member) / Lake, Douglas (Committee member) / Hinrichs, Richard (Committee member) / Arizona State University (Publisher)
Created2014
Description
Despite the approval of a Dengue virus (DV) vaccine in five endemic countries, dengue prevention would benefit from an immunization strategy highly immunogenic in young infants and not curtailed by viral interference. Problematically, infants younger than 9 year of age, whom are particularly prone to Dengue severe infection and death, cannot be immunized using current approved DV vaccine. The most important issues documented so far are the lack of efficiency and enhancement of the disease in young seronegative recipients, as well as uneven protection against the four DV serotypes. Based on data from clinical trials that showed enhanced performance of dengue vaccines when the host has previous anti-flaviviral immunity, I proposed here an attractive solution to complement the current vaccine: a recombinant measles vaccine vectoring dengue protective antigens to be administered to young infants. I hypothesized that recombinant measles virus expressing Dengue 2 and 4 antigens would successfully induce neutralizing responses against DV2 and 4 and the vaccine cocktail of this recombinant measles can prime anti-flaviviral neutralizing immunity. For this dissertation, I generated and performed preclinical immune assessment for four novel Measles-Dengue (MV-DV) vaccine candidates. I generated four MVs expressing the pre membrane (prM) and full length or truncated (90%) forms of the major envelope (E) from DV2 and DV4. Two virus, MVvac2-DV2(prME)N and MVvac2-DV4(prME), expressed high levels of membrane associated full-length E, while the other two viruses, MVvac2-DV2(prMEsol)N and MVvac2-DV4(prMEsol)N, expressed and secreted truncated, soluble E protein to its extracellular environment. The last two vectored vaccines proved superior anti-dengue neutralizing responses comparing to its corresponding full length vectors. Remarkably, when MVvac2-DV2/4(prMEsol)N recombinant vaccines were combined, the vaccine cocktail was able to prime cross-neutralizing responses against DV 1 and the relatively distant 17D yellow fever virus attenuated strain. Thus, I identify a promising DV vaccination strategy, MVvac2-DV2/4(prMEsol)N, which can prime broad neutralizing immune responses by using only two of the four available DV serotypes. The current MV immunization scheme can be advantageus to prime broad anti-flaviviral neutralizing immunity status, which will be majorly boosted by subsequent chimeric Dengue vaccine approaches.
ContributorsAbdelgalel, Rowida (Author) / Reyes del Valle, Jorge (Thesis advisor) / Mason, Hugh (Thesis advisor) / Lake, Douglas (Committee member) / Stout, Valerie (Committee member) / Frasch, Wayne (Committee member) / Arizona State University (Publisher)
Created2016
Description
Many Fic domain proteins, through catalyzing post translational modifications (PTM) of protein substrates, functionally contribute to bacterial pathogenesis and the regulation of bacterial growth. Furthermore, one form of Fic-mediated regulation is the Fic toxin-antitoxin system, whereby an antitoxin interacts with and inhibits the Fic toxin. This study sought to determine the functional importance of Mycobacterium tuberculosis Fic and its putative antitoxin protein, Rv3642c. Using M. tuberculosis H37Rv genetic deletion mutants, fic and Rv3642c were demonstrated to promote intracellular survival in human THP-1 macrophage-like cells. Unlike other Fic toxins, of Fic toxin-antitoxin systems, Fic did not inhibit bacterial growth in vitro in the absence of Rv3642c. Notably, Fic demonstrated in vitro AMPylation of a THP-1 cell extract protein as shown by immunodetection. Fic also exhibited auto-AMPylation activity. Interestingly, a mutation of the conserved histidine in the Fic domain motif, a residue previously shown to be critical for AMPylation, had no effect on Fic-mediated ATP hydrolysis or AMPylation activity. Rv3642c was demonstrated to form a complex with Fic when co-expressed in Escherichia coli, indicating a toxin-antitoxin interaction. Screening M. tuberculosis protein fractions and culture filtrate with α-Fic and α-Rv3642c rabbit antisera did not detect monomers of Fic or Rv3642c, thus the cellular localization of Fic and the Rv3642c-Fic complex remains unclear. The results of this study provide insight into the function of M. tuberculosis Fic, and suggest that Fic and Rv3642c are important for M. tuberculosis survival in the intracellular macrophage environment. Furthermore, these findings challenge the current dogma that Fic domain catalysis is dependent on the conserved histidine of the Fic motif.
ContributorsLaMarca, Ryan (Author) / Haydel, Shelley (Thesis advisor) / Lake, Douglas (Committee member) / Nickerson, Cheryl (Committee member) / Arizona State University (Publisher)
Created2017
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
Adoptive transfer of T cells engineered to express synthetic antigen-specific T cell receptors (TCRs) has provocative therapeutic applications for treating cancer. However, expressing these synthetic TCRs in a CD4+ T cell line is a challenge. The CD4+ Jurkat T cell line expresses endogenous TCRs that compete for space, accessory proteins, and proliferative signaling, and there is the potential for mixed dimer formation between the α and β chains of the endogenous receptor and that of the synthetic cancer-specific TCRs. To prevent hybridization between the receptors and to ensure the binding affinity measured with flow cytometry analysis is between the tetramer and the TCR construct, a CRISPR-Cas9 gene editing pipeline was developed. The guide RNAs (gRNAs) within the complex were designed to target the constant region of the α and β chains, as they are conserved between TCR clonotypes. To minimize further interference and confer cytotoxic capabilities, gRNAs were designed to target the CD4 coreceptor, and the CD8 coreceptor was delivered in a mammalian expression vector. Further, Golden Gate cloning methods were validated in integrating the gRNAs into a CRISPR-compatible mammalian expression vector. These constructs were transfected via electroporation into CD4+ Jurkat T cells to create a CD8+ knockout TCR Jurkat cell line for broadly applicable uses in T cell immunotherapies.
ContributorsHirneise, Gabrielle Rachel (Author) / Anderson, Karen (Thesis advisor) / Mason, Hugh (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2020
Description
Anoxia tolerance is strongly correlated with tolerance to heat, desiccation, hyperosmotic shock, freezing, and other general stressors, suggesting that anoxia tolerance is broadly related to stress tolerance. Age affects the capacity of many animals to survive anoxia, but the basis to this ontogenic variation is poorly understood. We exposed adult Drosophila, 1, 3, 5, 7, 9, and 12 days past eclosion, to six hours of anoxia and assessed survival 24-hours post-treatment. Survival of anoxia declined strongly with age (from 80% survival for one-day-old flies to 10% survival for 12 day-old-flies), a surprising result since adult fly senescence in Drosophila is usually observed much later. In anoxia, adenosine triphosphate (ATP) levels declined rapidly (< 30 min) to near-zero levels in both 1 and 12-day old adults; thus the higher anoxia-tolerance of young adults is not due to a better capacity to keep ATP elevated. Relatively few physiological parameters are reported to change over this age range in D. melanogaster, but gut bacterial content increases strongly. As a partial test for a causal link between bacterial load and anoxia tolerance, we replaced food daily, every third day, or every sixth day, and assayed survival of six hours of anoxia and bacterial load at 12 days of age. Anoxia tolerance for 12-day old flies was improved by more food changes and was strongly and negatively affected by bacterial load. These data suggest that increasing bacterial load may play an important role in the age-related decline of anoxia tolerance in Drosophila.
ContributorsSargent, James (Author) / Harrison, Jon F. (Thesis advisor) / Haydel, Shelly (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2020