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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
Infertility has become an increasing problem in developed countries and in many cases can be attributed to compromised sperm quality. Assessment of male fertility typically utilizes semen analysis which mainly examines sperm morphology, however many males whose sperm appear normal are sub- or infertile, suggesting that sperm from these males may be deficient in a protein or suite of proteins. To date, very little is known about the composition of sperm or the complex maturation process that confers motility and fertilization competency to sperm. Chapter 1 discusses the use of whole cell mass spectrometry to identify 1247 proteins comprising the Rhesus macaque (Macaca mulatta) sperm proteome, a commonly used model of human reproduction. This study provides a more robust proxy of human sperm composition than was previously available and facilitates studies of sperm using the rhesus macaque as a model. Chapters 2 & 3 provide a systems level overview of changes in sperm proteome composition that occurs during epididymal transit. Chapter 2 reports the proteomes of sperm collected from the caput, corpus and cauda segments of the mouse epididymis, identifying 1536, 1720 and 1234 proteins respectively. Chapter 3 reports the sperm proteome from four distinct segments of the Rhesus macaque epididymis, including the caput, proximal corpus, distal corpus and cauda, identifying 1951, 2014, 1764 and 1423 proteins respectively. These studies identify a number of proteins that are added and removed from sperm during epididymal transit which likely play an important role in the sperm maturation process. To date no comparative evolutionary studies of sperm proteomes have been undertaken. Chapter 4 compares four mammalian sperm proteomes including the human, macaque, mouse and rat. This study identified 98 proteins common to all four sperm proteomes, 82 primate and 90 rodent lineage-specific proteins and 494, 467, 566, and 193 species specific proteins in the human, macaque, mouse and rat sperm proteomes respectively and discusses how differences in sperm composition may ultimately lead to functional differences across species. Finally, chapter 5 uses sperm proteome data to inform the preliminary design of a rodent contraceptive vaccine delivered orally using recombinant attenuated Salmonella vaccine vectors.
ContributorsSkerget, Sheri Jo (Author) / Karr, Timothy L. (Thesis advisor) / Lake, Douglas (Committee member) / Petritis, Konstantinos (Committee member) / Arizona State University (Publisher)
Created2013
Description
Random peptide microarrays are a powerful tool for both the treatment and diagnostics of infectious diseases. On the treatment side, selected random peptides on the microarray have either binding or lytic potency against certain pathogens cells, thus they can be synthesized into new antimicrobial agents, denoted as synbodies (synthetic antibodies). On the diagnostic side, serum containing specific infection-related antibodies create unique and distinct "pathogen-immunosignatures" on the random peptide microarray distinct from the healthy control serum, and this different mode of binding can be used as a more precise measurement than traditional ELISA tests. My thesis project is separated into these two parts: the first part falls into the treatment side and the second one focuses on the diagnostic side. My first chapter shows that a substitution amino acid peptide library helps to improve the activity of a recently reported synthetic antimicrobial peptide selected by the random peptide microarray. By substituting one or two amino acids of the original lead peptide, the new substitutes show changed hemolytic effects against mouse red blood cells and changed potency against two pathogens: Staphylococcus aureus and Pseudomonas aeruginosa. Two new substitutes are then combined together to form the synbody, which shows a significantly antimicrobial potency against Staphylococcus aureus (<0.5uM). In the second chapter, I explore the possibility of using the 10K Ver.2 random peptide microarray to monitor the humoral immune response of dengue. Over 2.5 billion people (40% of the world's population) live in dengue transmitting areas. However, currently there is no efficient dengue treatment or vaccine. Here, with limited dengue patient serum samples, we show that the immunosignature has the potential to not only distinguish the dengue infection from non-infected people, but also the primary dengue infection from the secondary dengue infections, dengue infection from West Nile Virus (WNV) infection, and even between different dengue serotypes. By further bioinformatic analysis, we demonstrate that the significant peptides selected to distinguish dengue infected and normal samples may indicate the epitopes responsible for the immune response.
ContributorsWang, Xiao (Author) / Johnston, Stephen Albert (Thesis advisor) / Blattman, Joseph (Committee member) / Arntzen, Charles (Committee member) / Arizona State University (Publisher)
Created2013
Description
Coccidioidomycosis, also known as Valley Fever, is a disease caused by the dimorphic soil-dwelling fungus, Coccidioides sp. Coccidioidomycosis is difficult to diagnose because symptoms are similar to community-acquired pneumonia. Current diagnostic tests rely on antibody responses, but immune responses can be delayed and aberrant, resulting in false negative diagnoses. Unlike serology, detection of coccidioidal proteins or other fungal components in blood could distinguish valley fever from other pulmonary infections and provide a definitive diagnosis. Using mass spectrometry (LC-MS/MS) we examined the plasma peptidome from patients with serologically confirmed coccidioidomycosis. Mass spectra were searched using the protein database from the Coccidioides species, generated and annotated by the Broad Institute. 15 of 20 patients with serologically confirmed coccidioidomycosis demonstrated the presence of a peptide in plasma, "PGLDSKSLACTFSQV" (PGLD). The peptide is derived from an open reading frame from a "conserved hypothetical protein" annotated with 2 exons, and to date, found only in the C. posadasii strain Silviera RMSCC 3488 genomic sequence. In this thesis work, cDNA sequence analysis from polyadenylated RNA confirms the peptide sequence and genomic location of the peptide, but does not indicate that the intron in the gene prediction of C. posadasii strain Silviera RMSCC 3488 is present. A monoclonal antibody generated against the peptide bound to a 16kDa protein in T27K coccidioidal lysate. Detecting components of the fungus plasma could be a useful diagnostic tool, especially when serology does not provide a definitive diagnosis.
ContributorsDuffy, Stacy Leigh (Author) / Lake, Douglas (Thesis advisor) / Magee, Dewey Mitch (Committee member) / Antwi, Kwasi (Committee member) / Arizona State University (Publisher)
Created2013
Description
Background: Coccidioidomycosis (Valley Fever) is a respiratory disease that is caused by the soil-dwelling fungi Coccidioides immitis and Coccidioides posadasii. Because fungal glycosylation patterns are distinct from mammalian glycosylation patterns, we hypothesized that certain lectins (carbohydrate-binding proteins) might have differential binding properties to coccidioidal glycoproteins, and therefore serve as a tool for the purification and characterization of these glycoproteins from patient specimens. Materials and Methods: To identify potential Coccidioides-binding lectins, lectin-based immunohistochemistry was performed using a panel of 21 lectins on lung tissue from human patients infected with Coccidioides. Enzyme-Linked Immunosorbent Assays (ELISAs) were used to confirm and test candidate Coccidioides-binding lectins for their ability to bind to proteins from antigen preparations of laboratory-grown Coccidioides. Inhibition IHC and ELISAs were used to confirm binding properties of these lectins. SDS-PAGE and mass spectrometry were performed on eluates from coccidioidal antigen preparations run through lectin-affinity chromatography columns to characterize and identify lectin-binding coccidioidal glycoproteins. Results: Two GlcNAc-binding lectins, GSLII and sWGA, bound specifically to spherules and endospores in infected human lung tissue, and not to adjacent lung tissue. The binding of these lectins to both Coccidioides proteins in lung tissue and to coccidioidal antigen preparations was confirmed to have lectin-like characteristics. SDS-PAGE analysis of eluates from lectin-affinity chromatography demonstrated that GSLII and sWGA bind to coccidioidal glycoproteins. Mass spectrometric identification of the top ten lectin affinity-purified glycoproteins demonstrated that GSLII and sWGA share affinity to a common set of coccidioidal glycoproteins. Conclusion: This is the first report of lectins that bind specifically to Coccidioides spherules and endospores in infected humans. These lectins may have the potential to serve as tools for a better method of detection and diagnosis of Valley Fever.
ContributorsChowdhury, Yasmynn (Author) / Lake, Douglas (Thesis director) / Grys, Thomas (Committee member) / Magee, Mitchell (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of Human Evolution and Social Change (Contributor)
Created2015-05
Description
Human Immunodeficiency Virus type 1 (HIV-1) causes millions of deaths every year, but a protective vaccine remains elusive. A promising vaccine strategy is to use virus-like particles (VLPs) for HIV-1. To this end, HIV-1 VLPs were produced in Nicotiana benthamiana plants that were stably expressing the HIV-1 Gag protein and transiently expressing a truncated form of gp41. These VLPs were tested to determine their inherent adjuvant effects due to their production in plants in order to dissect the previously observed stimulating activity of these VLPs in a prime-boost vaccine approach. THP1 human monocytes were differentiated using PMA or IL-4 and GM-CSF to form macrophages and dendritic cells, respectively. These cells were treated with purified VLPs or control samples to determine the individual adjuvant effects of the plant, bacterial, and VLP components in the purified VLP samples. It was postulated that the PMA-differentiated THP1 cells were not induced to become macrophages due to the lack of CD11b+ cells in the sample and the lack of increased TNFα expression in response to LPS treatment. It was also determined that the VLPs have inherent adjuvant properties to dendritic cells due to bacterial and VLP components, but not due to plant components.
ContributorsDickey, Rebekah Marie (Author) / Mor, Tsafrir (Thesis director) / Blattman, Joseph (Committee member) / Meador, Lydia (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Non-small cell lung cancer (NSCLC) has become the leading cause of cancer-related deaths in the United States with a combined 5-year survival rate of only 16%. Even with advancements in aggressive chemotherapeutics, there has been little improvement in patient survival. LKB1 (liver kinase B1)/STK11 (serine-threonine kinase 11) is a tumor suppressor gene mutated in ~30% of NSCLC adenocarcinomas and loss of LKB1 is associated with a more aggressive cancer phenotype. In LKB1-deficient NSCLC, we observe significantly elevated expression and secretion of the chemokines CCL2, CCL5, and CCL20, which are involved in macrophage recruitment. Numerous studies have shown that high infiltration of a unique subset of macrophages called tumor-associated macrophages (TAMs) is associated with poor prognosis in patients with various cancers. mTORC1-HIF1-α and NFκB are two pathways that have been shown to regulate chemokine secretion and are often up-regulated in the absence of LKB1. Dosing LKB1-null cell lines with inhibitors of mTOR and NFκB in addition to silencing HIF1-α gene expression demonstrate that NFκB but not mTORC1-HIF1-α signaling may play a role in regulating chemokine secretion in LKB1-deficient NSCLC. Collectively, these results provide insight into the mechanisms responsible for the aggressive phenotype associated with LKB1-deficient non-small cell lung cancer.
ContributorsO'Brien, Kelley Xiao-Fung (Author) / Blattman, Joseph (Thesis director) / Inge, Landon (Committee member) / Friel, Jacqueline (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Among wild rodent populations, vertical transmission is believed to constitute the primary route of infection for Lymphocytic Choriomeningitis Virus (LCMV), a non-lytic arenavirus with both acute and chronic forms. When carrier mice infected at birth with the acute Armstrong strain reproduce, they generate congenital carrier offspring containing a quasispecies of LCMV that includes Armstrong as well as its chronic Clone-13 variant. This study examined the genetic trends in the vertical transmission of LCMV from mothers infected perinatally with Clone-13. Viral isolates obtained from the serum of congenital carrier offspring were partially sequenced to reveal residue 260 in the glycoprotein-encoding region of their S segment, the site of a major amino acid change differentiating the chronic and acute strains. It was found that the phenylalanine-to-leucine mutation associated with Clone-13 was present in 100% of the isolates, strongly indicating that the offspring of Clone-13 carriers contain exclusively the chronic variant. This research has broad implications for the epidemiology of the virus, and, given the predominance of Armstrong in the wild, suggests that there must be a biological cost associated with Clone-13 infection in non-carriers.
ContributorsFrear, Cody Christian (Author) / Blattman, Joseph (Thesis director) / Hogue, Brenda (Committee member) / Holechek, Susan (Committee member) / Barrett, The Honors College (Contributor) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Efforts to quantify the diversity of the T cell repertoire have generally been unsuccessful because not all factors accounting for diversity have been considered. In order to get an accurate representation of the T cell repertoire, one must incorporate analysis of germline gene diversity, diversity from somatic recombination, joining diversity from N- and P- nucleotides, and TCR chain pairing diversity. Because of advances in high-throughput sequencing techniques, estimates have been able to account for diversity from TCR genes. However the ability to account for chain pairing diversity has been more difficult. In order to do so, single cell sorting techniques must be employed. These techniques, though effective, are time consuming and expensive. For this reason, no large-scale analyses have been done on the immune repertoires using these techniques. In this study, we propose a novel method for linking the two TCR chain sequences from an individual cell. DNA origami nanostructure technology is employed to capture and bind the TCRγ and TCRδ chain mRNA inside individual cells using probe strands complementary to the C-region of those sequences. We then use a dual-primer RT and ligation molecular strategy to link the two sequences together. The result is a single amplicon containing the CDR3 region of the TCRγ and TCRδ. This amplicon can then be easily PCR amplified using sequence specific primers, and sequenced. DNA origami nanostructures offer a rapid, cost-effective method alternative to conventional single cell sorting techniques, as both TCR mRNA can be captured on one origami molecule inside a single cell. At present, this study outlines a proof-of-principle analysis of the method to determine its functionality. Using known TCRγ and TCRδ sequences, the DNA origami and RT/PCR method was tested and resulting sequence data proved the effectiveness of the method. The original TCRγ and TCRδ sequences were linked together as a single amplicon containing both CDR3 regions of the genes. Thus, this method can be employed in further research to elucidate the γδ T cell repertoire. This technology is also easily adapted to any gene target or cell type and therefore presents a large opportunity to be used in other immune repertoire analysis and other immunological studies (such as the rapid identification and subsequent production of antibodies).
ContributorsPoindexter, Morgan Elizabeth (Author) / Blattman, Joseph (Thesis director) / Yan, Hao (Committee member) / Schoettle, Louis (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Patients who are infected with the human immunodeficiency virus (HIV) and who remain adherent to their highly active antiretroviral treatment (HAART) regimen are likely to achieve good virologic control over significant periods of time. Children who start with a low CD4 percentage (below 15%) are associated with adverse clinical outcomes and the risk of never increasing their CD4 counts to normal functioning levels. While adherent adult HIV patients have been studied frequently, this was a retrospective chart study that aimed to describe the immune reconstitution pattern for up to 16 years in virologically controlled pediatric patients who had been or who are currently being treated at the Bill Holt Clinic at Phoenix Children's Hospital. In the preliminary study, 35 patients met criteria for inclusion and three years later for this extension study 7 more were added while 17 of the initial patients were followed further because they have remained in care and virologically controlled. All 28 patients who achieved 5 years of viral suppression were >25% CD4. All 8 patients who achieved 12 years of viral suppression were >31% CD4. All patients who achieved 16 years of viral suppression were >41% CD4. After 12 years, the 8 patients who maintained viral suppression all had absolute CD4 counts of over 600 cells and additionally each had CD4/CD8 ratios greater than 1. Overall, the data shows immune system normalization for up to 16 years, although CD4/CD8 ratios improved but never completely normalized. Some limitations include a small sample size and missing data points due to laboratory testing errors or the lack of technology in different countries to test for CD8 cells. These findings suggest that children who remain adherent to HAART can experience ongoing immune healing for up to 16 years. This may provide additional incentive to providers and caretakers to encourage adherence and maximize long-term immune competence in HIV positive children.
ContributorsRichards, Anne Elizabeth (Author) / Wachter, Rebekka (Thesis director) / Blattman, Joseph (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Human Evolution and Social Change (Contributor)
Created2015-05