Matching Items (141)
Description
The majority of chronic myeloid leukemia (CML) and some of acute lymphocytic leukemia (ALL) cases are associated with possessing the BCR-Abl fusion protein from an oncogenic translocation, resulting in a constantly active form of Abl and rapid proliferation. CML and ALL cells that possess the BCR-Abl fusion protein are known as Philadelphia chromosome positive (Ph+). Currently, Imatinib (selective Abl inhibitor) is used as therapy against CML and ALL. However, some patients may have malignancies which show resistance to Imatinib. Previous work displays that the transformation of progenitor B cells with the v-Abl oncogene of Abelson murine leukemia virus results in cell cycle progression, rapid proliferation, and potentially malignant transformation while preventing any further differentiation. Progenitor B cells transformed with the temperature-sensitive form of the v-Abl oncogene have served as a model to study cellular response to Imatinib treatment. After some manipulation, very few cells were forced to progress to malignancy, forming tumor in vivo. These cells were no long sensitive to v-Abl inactivation, resembling the Imatinib resistant ALL. Autophagy is the process by which proteins and organelles are broken-down and recycled within the eukaryotic cell and has been hypothesized to play a part in cancer cell survival and drug-resistance. LC3 processing is a widely accepted marker of autophagy induction and progression. It has also been shown that Imatinib treatment of Ph+ leukemia can induce autophagy. In this study, we examined the autophagy induction in response to v-Abl inactivation in a Ph+-B-ALL cell model that shows resistance to Imatinib. In particular, we wonder whether the tumor cell line resistant to v-Abl inactivation may acquire a high level of autophagy to become resistant to apoptosis induced by v-Abl inactivation, and thus become addicted to autophagy. Indeed, this tumor cell line displays a high basal levels of LC3 I and II expression, regardless of v-Abl activity. We further demonstrated that inhibition of the autophagy pathway enhances the tumor line's sensitivity to Imatinib, resulting in cell cycle arrest and massive apoptosis. The combination of autophagy and Abl inhibitions may serve as an effective therapy for BCR-Abl positive CML.
ContributorsArkus, Nohea (Author) / Chang, Yung (Thesis advisor) / Kusumi, Kenro (Committee member) / Lake, Douglas (Committee member) / Jacobs, Bertram (Committee member) / Arizona State University (Publisher)
Created2011
Description
A synbody is a newly developed protein binding peptide which can be rapidly produced by chemical methods. The advantages of the synbody producing process make it a potential human proteome binding reagent. Most of the synbodies are designed to bind to specific proteins. The peptides incorporated in a synbody are discovered with peptide microarray technology. Nevertheless, the targets for unknown synbodies can also be discovered by searching through a protein mixture. The first part of this thesis mainly focuses on the process of target searching, which was performed with immunoprecipitation assays and mass spectrometry analysis. Proteins are pulled down from the cell lysate by certain synbodies, and then these proteins are identified using mass spectrometry. After excluding non-specific bindings, the interaction between a synbody and its real target(s) can be verified with affinity measurements. As a specific example, the binding between 1-4-KCap synbody and actin was discovered. This result proved the feasibility of the mass spectrometry based method and also suggested that a high throughput synbody discovery platform for the human proteome could be developed. Besides the application of synbody development, the peptide microarray technology can also be used for immunosignatures. The composition of all types of antibodies existing in one's blood is related to an individual's health condition. A method, called immunosignaturing, has been developed for early disease diagnosis based on this principle. CIM10K microarray slides work as a platform for blood antibody detection in immunosignaturing. During the analysis of an immunosignature, the data from these slides needs to be validated by using landing light peptides. The second part of this thesis focuses on the validation of the data. A biotinylated peptide was used as a landing light on the new CIM10K slides. The data was collected in several rounds of tests and indicated that the variation among landing lights was significantly reduced by using the newly prepared biotinylated peptide compared with old peptide mixture. Several suggestions for further landing light improvement are proposed based on the results.
ContributorsSun, Minyao (Author) / Johnston, Stephen Albert (Thesis advisor) / Diehnelt, Chris Wayne (Committee member) / Stafford, Phillip (Committee member) / Arizona State University (Publisher)
Created2011
Description
Immunosignaturing is a new immunodiagnostic technology that uses random-sequence peptide microarrays to profile the humoral immune response. Though the peptides have little sequence homology to any known protein, binding of serum antibodies may be detected, and the pattern correlated to disease states. The aim of my dissertation is to analyze the factors affecting the binding patterns using monoclonal antibodies and determine how much information may be extracted from the sequences. Specifically, I examined the effects of antibody concentration, competition, peptide density, and antibody valence. Peptide binding could be detected at the low concentrations relevant to immunosignaturing, and a monoclonal's signature could even be detected in the presences of 100 fold excess naive IgG. I also found that peptide density was important, but this effect was not due to bivalent binding. Next, I examined in more detail how a polyreactive antibody binds to the random sequence peptides compared to protein sequence derived peptides, and found that it bound to many peptides from both sets, but with low apparent affinity. An in depth look at how the peptide physicochemical properties and sequence complexity revealed that there were some correlations with properties, but they were generally small and varied greatly between antibodies. However, on a limited diversity but larger peptide library, I found that sequence complexity was important for antibody binding. The redundancy on that library did enable the identification of specific sub-sequences recognized by an antibody. The current immunosignaturing platform has little repetition of sub-sequences, so I evaluated several methods to infer antibody epitopes. I found two methods that had modest prediction accuracy, and I developed a software application called GuiTope to facilitate the epitope prediction analysis. None of the methods had sufficient accuracy to identify an unknown antigen from a database. In conclusion, the characteristics of the immunosignaturing platform observed through monoclonal antibody experiments demonstrate its promise as a new diagnostic technology. However, a major limitation is the difficulty in connecting the signature back to the original antigen, though larger peptide libraries could facilitate these predictions.
ContributorsHalperin, Rebecca (Author) / Johnston, Stephen A. (Thesis advisor) / Bordner, Andrew (Committee member) / Taylor, Thomas (Committee member) / Stafford, Phillip (Committee member) / Arizona State University (Publisher)
Created2011
Description
African Swine Fever (ASF), endemic in many African countries, is now spreading to other continents. Though ASF is capable of incurring serious economic losses in affected countries, no vaccine exists to provide immunity to animals. Disease control relies largely on rapid diagnosis and the implementation of movement restrictions and strict eradication programs. Developing a scalable, accurate and low cost diagnostic for ASF will be of great help for the current situation. CIM's 10K random peptide microarray is a new high-throughput platform that allows systematic investigations of immune responses associated with disease and shows promise as a diagnostic tool. In this study, this new technology was applied to characterize the immune responses of ASF virus (ASFV) infections and immunizations. Six sets of sera from ASFV antigen immunized pigs, 6 sera from infected pigs and 20 sera samples from unexposed pigs were tested and analyzed statistically. Results show that both ASFV antigen immunized pigs and ASFV viral infected pigs can be distinguished from unexposed pigs. Since it appears that immune responses to other viral infections are also distinguishable on this platform, it holds the potential of being useful in developing a new ASF diagnostic. The ability of this platform to identify specific ASFV antibody epitopes was also explored. A subtle motif was found to be shared among a set of peptides displaying the highest reactivity for an antigen specific antibody. However, this motif does not seem to match with any antibody epitopes predicted by a linear antibody epitope prediction.
ContributorsXiao, Liang (Author) / Sykes, Kathryn (Thesis advisor) / Zhao, Zhan-Gong (Committee member) / Stafford, Phillip (Committee member) / Arizona State University (Publisher)
Created2011
Description
Pathogenic Gram-negative bacteria employ a variety of molecular mechanisms to combat host defenses. Two-component regulatory systems (TCR systems) are the most ubiquitous signal transduction systems which regulate many genes required for virulence and survival of bacteria. In this study, I analyzed different TCR systems in two clinically-relevant Gram-negative bacteria, i.e., oral pathogen Porphyromonas gingivalis and enterobacterial Escherichia coli. P. gingivalis is a major causative agent of periodontal disease as well as systemic illnesses, like cardiovascular disease. A microarray study found that the putative PorY-PorX TCR system controls the secretion and maturation of virulence factors, as well as loci involved in the PorSS secretion system, which secretes proteinases, i.e., gingipains, responsible for periodontal disease. Proteomic analysis (SILAC) was used to improve the microarray data, reverse-transcription PCR to verify the proteomic data, and primer extension assay to determine the promoter regions of specific PorX regulated loci. I was able to characterize multiple genetic loci regulated by this TCR system, many of which play an essential role in hemagglutination and host-cell adhesion, and likely contribute to virulence in this bacterium. Enteric Gram-negative bacteria must withstand many host defenses such as digestive enzymes, low pH, and antimicrobial peptides (AMPs). The CpxR-CpxA TCR system of E. coli has been extensively characterized and shown to be required for protection against AMPs. Most recently, this TCR system has been shown to up-regulate the rfe-rff operon which encodes genes involved in the production of enterobacterial common antigen (ECA), and confers protection against a variety of AMPs. In this study, I utilized primer extension and DNase I footprinting to determine how CpxR regulates the ECA operon. My findings suggest that CpxR modulates transcription by directly binding to the rfe promoter. Multiple genetic and biochemical approaches were used to demonstrate that specific TCR systems contribute to regulation of virulence factors and resistance to host defenses in P. gingivalis and E. coli, respectively. Understanding these genetic circuits provides insight into strategies for pathogenesis and resistance to host defenses in Gram negative bacterial pathogens. Finally, these data provide compelling potential molecular targets for therapeutics to treat P. gingivalis and E. coli infections.
ContributorsLeonetti, Cori (Author) / Shi, Yixin (Thesis advisor) / Stout, Valerie (Committee member) / Nickerson, Cheryl (Committee member) / Sandrin, Todd (Committee member) / Arizona State University (Publisher)
Created2013
Description
The study of bacterial resistance to antimicrobial peptides (AMPs) is a significant area of interest as these peptides have the potential to be developed into alternative drug therapies to combat microbial pathogens. AMPs represent a class of host-mediated factors that function to prevent microbial infection of their host and serve as a first line of defense. To date, over 1,000 AMPs of various natures have been predicted or experimentally characterized. Their potent bactericidal activities and broad-based target repertoire make them a promising next-generation pharmaceutical therapy to combat bacterial pathogens. It is important to understand the molecular mechanisms, both genetic and physiological, that bacteria employ to circumvent the bactericidal activities of AMPs. These understandings will allow researchers to overcome challenges posed with the development of new drug therapies; as well as identify, at a fundamental level, how bacteria are able to adapt and survive within varied host environments. Here, results are presented from the first reported large scale, systematic screen in which the Keio collection of ~4,000 Escherichia coli deletion mutants were challenged against physiologically significant AMPs to identify genes required for resistance. Less than 3% of the total number of genes on the E. coli chromosome was determined to contribute to bacterial resistance to at least one AMP analyzed in the screen. Further, the screen implicated a single cellular component (enterobacterial common antigen, ECA) and a single transporter system (twin-arginine transporter, Tat) as being required for resistance to each AMP class. Using antimicrobial resistance as a tool to identify novel genetic mechanisms, subsequent analyses were able to identify a two-component system, CpxR/CpxA, as a global regulator in bacterial resistance to AMPs. Multiple previously characterized CpxR/A members, as well as members found in this study, were identified in the screen. Notably, CpxR/A was found to transcriptionally regulate the gene cluster responsible for the biosynthesis of the ECA. Thus, a novel genetic mechanism was uncovered that directly correlates with a physiologically significant cellular component that appears to globally contribute to bacterial resistance to AMPs.
ContributorsWeatherspoon-Griffin, Natasha (Author) / Shi, Yixin (Thesis advisor) / Clark-Curtiss, Josephine (Committee member) / Misra, Rajeev (Committee member) / Nickerson, Cheryl (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2013
Description
Well-established model systems exist in four out of the seven major classes of vertebrates. These include the mouse, chicken, frog and zebrafish. Noticeably missing from this list is a reptilian model organism for comparative studies between the vertebrates and for studies of biological processes unique to reptiles. To help fill in this gap the green anole lizard, Anolis carolinensis, is being adapted as a model organism. Despite the recent release of the complete genomic sequence of the A. carolinensis, the lizard lacks some resources to aid researchers in their studies. Particularly, the lack of transcriptomic resources for lizard has made it difficult to identify genes complete with alternative splice forms and untranslated regions (UTRs). As part of this work the genome annotation for A. carolinensis was improved through next generation sequencing and assembly of the transcriptomes from 14 different adult and embryonic tissues. This revised annotation of the lizard will improve comparative studies between vertebrates, as well as studies within A. carolinensis itself, by providing more accurate gene models, which provide the bases for molecular studies. To demonstrate the utility of the improved annotations and reptilian model organism, the developmental process of somitogenesis in the lizard was analyzed and compared with other vertebrates. This study identified several key features both divergent and convergent between the vertebrates, which was not previously known before analysis of a reptilian model organism. The improved genome annotations have also allowed for molecular studies of tail regeneration in the lizard. With the annotation of 3' UTR sequences and next generation sequencing, it is now possible to do expressional studies of miRNA and predict their mRNA target transcripts at genomic scale. Through next generation small RNA sequencing and subsequent analysis, several differentially expressed miRNAs were identified in the regenerating tail, suggesting miRNA may play a key role in regulating this process in lizards. Through miRNA target prediction several key biological pathways were identified as potentially under the regulation of miRNAs during tail regeneration. In total, this work has both helped advance A. carolinensis as model system and displayed the utility of a reptilian model system.
ContributorsEckalbar, Walter L (Author) / Kusumi, Kenro (Thesis advisor) / Huentelman, Matthew (Committee member) / Rawls, Jeffery (Committee member) / Wilson-Rawls, Norma (Committee member) / Arizona State University (Publisher)
Created2012
Description
Induced pluripotent stem cells (iPSCs) are an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects. Postmortem histopathological examination of a donor's brain, combined with premortem clinical criteria, is often the most robust approach to correctly classify an individual as a disease-specific case or unaffected control. We describe the establishment of primary dermal fibroblasts cells lines from 28 autopsy donors. These fibroblasts were used to examine the proliferative effects of establishment protocol, tissue amount, biopsy site, and donor age. As proof-of-principle, iPSCs were generated from fibroblasts from a 75-year-old male, whole body donor, defined as an unaffected neurological control by both clinical and histopathological criteria. To our knowledge, this is the first study describing autopsy donor-derived somatic cells being used for iPSC generation and subsequent neural differentiation. This unique approach also enables us to compare iPSC-derived cell cultures to endogenous tissues from the same donor. We utilized RNA sequencing (RNA-Seq) to evaluate the transcriptional progression of in vitro-differentiated neural cells (over a timecourse of 0, 35, 70, 105 and 140 days), and compared this with donor-identical temporal lobe tissue. We observed in vitro progression towards the reference brain tissue, supported by (i) a significant increasing monotonic correlation between the days of our timecourse and the number of actively transcribed protein-coding genes and long intergenic non-coding RNAs (lincRNAs) (P < 0.05), consistent with the transcriptional complexity of the brain, (ii) an increase in CpG methylation after neural differentiation that resembled the epigenomic signature of the endogenous tissue, and (iii) a significant decreasing monotonic correlation between the days of our timecourse and the percent of in vitro to brain-tissue differences (P < 0.05) for tissue-specific protein-coding genes and all putative lincRNAs. These studies support the utility of autopsy donors' somatic cells for iPSC-based neurological disease models, and provide evidence that in vitro neural differentiation can result in physiologically progression.
ContributorsHjelm, Brooke E (Author) / Craig, David W. (Thesis advisor) / Wilson-Rawls, Norma J. (Thesis advisor) / Huentelman, Matthew J. (Committee member) / Mason, Hugh S. (Committee member) / Kusumi, Kenro (Committee member) / Arizona State University (Publisher)
Created2013
Description
The development of the vertebrate musculoskeletal system is a highly dynamic process, requiring tight control of the specification and patterning of myogenic, chondrogenic and tenogenic cell types. Development of the diverse musculoskeletal lineages from a common embryonic origin in the paraxial mesoderm indicates the presence of a regulatory network of transcription factors that direct lineage decisions. The basic helix-loop-helix transcription factor, PARAXIS, is expressed in the paraxial mesoderm during vertebrate somitogenesis, where it has been shown to play a critical role in the mesenchymal-to-epithelial transition associated with somitogenesis, and the development of the hypaxial skeletal musculature and axial skeleton. In an effort to elucidate the underlying genetic mechanism by which PARAXIS regulates the musculoskeletal system, I performed a microarray-based, genome-wide analysis comparing transcription levels in the somites of Paraxis-/- and Paraxis+/+ embryos. This study revealed targets of PARAXIS involved in multiple aspects of mesenchymal-to-epithelial transition, including Fap and Dmrt2, which modulate cell-extracellular matrix adhesion. Additionally, in the epaxial dermomyotome, PARAXIS activates the expression of the integrin subunits a4 and a6, which bind fibronectin and laminin, respectively, and help organize the patterning of trunk skeletal muscle. Finally, PARAXIS activates the expression of genes required for the epithelial-to-mesenchymal transition and migration of hypaxial myoblasts into the limb, including Lbx1 and Met. Together, these data point to a role for PARAXIS in the morphogenetic control of musculoskeletal patterning.
ContributorsRowton, Megan (Author) / Rawls, Alan (Thesis advisor) / Wilson-Rawls, Jeanne (Committee member) / Kusumi, Kenro (Committee member) / Gadau, Juergen (Committee member) / Arizona State University (Publisher)
Created2013
Description
Vertebrate genomes demonstrate a remarkable range of sizes from 0.3 to 133 gigabase pairs. The proliferation of repeat elements are a major genomic expansion. In particular, long interspersed nuclear elements (LINES) are autonomous retrotransposons that have the ability to "cut and paste" themselves into a host genome through a mechanism called target-primed reverse transcription. LINES have been called "junk DNA," "viral DNA," and "selfish" DNA, and were once thought to be parasitic elements. However, LINES, which diversified before the emergence of many early vertebrates, has strongly shaped the evolution of eukaryotic genomes. This thesis will evaluate LINE abundance, diversity and activity in four anole lizards. An intrageneric analysis will be conducted using comparative phylogenetics and bioinformatics. Comparisons within the Anolis genus, which derives from a single lineage of an adaptive radiation, will be conducted to explore the relationship between LINE retrotransposon activity and causal changes in genomic size and composition.
ContributorsMay, Catherine (Author) / Kusumi, Kenro (Thesis advisor) / Gadau, Juergen (Committee member) / Rawls, Jeffery A (Committee member) / Arizona State University (Publisher)
Created2013