Matching Items (64)
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
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
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
The long-term survival of patients with glioblastoma multiforme is compromised by the tumor's proclivity for local invasion into the surrounding normal brain. These invasive cells escape surgery and display resistance to chemotherapeutic- and radiation-induced apoptosis. We have previously shown that tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell invasion and survival via binding to the fibroblast growth factor-inducible 14 (Fn14) receptor and subsequent activation of the Rac1/NF-kappaB pathway. In addition, we have reported previously that Fn14 is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Here we demonstrate that TWEAK can act as a chemotactic factor for glioma cells, a potential process to drive cell invasion into the surrounding brain tissue. Specifically, we detected a chemotactic migration of glioma cells to the concentration gradient of TWEAK. Since Src family kinases (SFK) have been implicated in chemotaxis, we next determined whether TWEAK:Fn14 engagement activated these cytoplasmic tyrosine kinases. Our data shows that TWEAK stimulation of glioma cells results in a rapid phosphorylation of the SFK member Lyn as determined by multiplex Luminex assay and verified by immunoprecipitation. Immunodepletion of Lyn by siRNA oligonucleotides suppressed the chemoattractive effect of TWEAK on glioma cells. We hypothesize that TWEAK secretion by cells present in the glioma microenvironment induce invasion of glioma cells into the brain parenchyma. Understanding the function and signaling of the TWEAK-Fn14 ligand-receptor system may lead to development of novel therapies to therapeutically target invasive glioma cells.
ContributorsJameson, Nathan Meade (Author) / Anderson, Karen (Thesis director) / Lake, Douglas (Committee member) / Tran, Nhan (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
Oropharyngeal cancer (OPC) is the world's sixth most common cancer and in many cases is associated with infection with human papillomavirus (HPV) type 16. Antibodies (Abs) to HPV16 viral antigens are potential diagnostic biomarkers of HPV-associated OPC (HPV OPC). A custom multiplexed bead array assay was used to detect Abs to HPV16 antigens E1, CE2, NE2, E4, E5, E6, E7, L1, and L2. Following extensive optimization of the assay, these genes were expressed as GST-fusion proteins and captured onto anti-GST magnetic beads. Serum was obtained from 256 OPC patients at the time of diagnosis and from 78 healthy controls. The median fluorescent intensity (MFI) was determined for each antigen and ratios of MFI to control GST-fusion protein were determined for each serum sample. Cutoff values were set as the mean + 3 SD of the MFIs of healthy controls and p-values were calculated using Wilcoxon unpaired and Fisher's exact test. Results of this experiment showed that HPV16 E1, CE2, NE2, E4, E6, and E7 Ab levels were elevated in OPC patients compared to controls (p<0.001), as were Ab levels to L1 (p = 0.013) and L2 (p = 0.023), per Fischer's exact test. Abs to CE2, NE2, E6, and E7 were identified as a potential biomarker panel for early detection of HPV OPC. For the 111 patients with known HPV+ tumors as measured by tumor PCR of E6 and/or E7, this assay had a sensitivity of 90% and specificity of 87% (AUC = 0.96). From these results, we conclude that custom bead array assays can be used to detect HPV16 Abs in patient sera, and we have identified a 4-Ab biomarker panel for the early detection of HPV OPC.
ContributorsGoulder, Alison Leigh (Author) / Anderson, Karen (Thesis director) / Lake, Douglas (Committee member) / Cheng, Julia (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
Cancer is one of the leading causes of death in the world and represents a tremendous burden on patients, families and societies. S. Typhimurium strains are specifically attracted to compounds produced by cancer cells and could overcome the traditional therapeutic barrier. However, a major problem with using live attenuated Salmonella as anti-cancer agents is their toxicity at the dose required for therapeutic efficacy, but reducing the dose results in diminished efficacy. In this project, we explored novel means to reduce the toxicity of the recombinant attenuated Salmonella by genetically engineering those virulence factors to facilitate maximal colonization of tumor tissues and reduced fitness in normal tissues. We have constructed two sets of Salmonella strains. In the first set, each targeted gene was knocked out by deletion of the gene. In the second set, the predicted promoter region of each gene was replaced with a rhamnose-regulated promoter, which will cease the synthesis of these genes in vivo, a rhamnose-free environment.
ContributorsBenson, Lee Samuel (Author) / Kong, Wei (Thesis director) / Martin, Thomas (Committee member) / Lake, Douglas (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor) / Center for Infectious Diseases and Vaccinology (Contributor) / School of Life Sciences (Contributor)
Created2013-05
DescriptionA novel and unconventional approach for delivering a eukaryotic apoptosis factor, TNF-related apoptosis-inducing ligand (TRAIL), to cancer cells within and around necrotizing tumors by utilizing a S. Typhimurium purine requiring auxotroph as a biological vector to develop two anticancer therapies with multiple modality and broad economic feasibility.
ContributorsKoons, Andrew (Author) / Curtiss, Roy (Thesis director) / Lake, Douglas (Committee member) / Janthakahalli, Nagaraj Vinay (Committee member) / Barrett, The Honors College (Contributor)
Created2013-12
Description
While the entire human genome has been sequenced, the understanding of its functional elements remains unclear. The Encyclopedia of DNA Elements (ENCODE) project analyzed 1% of the human genome and found that the majority of the human genome is transcribed, including non-protein coding regions. The hypothesis is that some of the "non-coding" sequences are translated into peptides and small proteins. Using mass spectrometry numerous peptides derived from the ENCODE transcriptome were identified. Peptides and small proteins were also found from non-coding regions of the 1% of the human genome that the ENCODE did not find transcripts for. A large portion of these peptides mapped to the intronic regions of known genes, thus it is suspected that they may be undiscovered exons present in alternative spliceoforms of certain genes. Further studies proved the existence of polyadenylated RNAs coding for these peptides. Although their functional significance has not been determined, I anticipate the findings will lead to the discovery of new splice variants of known genes and possibly new transcriptional and translational mechanisms.
ContributorsWang, Lulu (Author) / Lake, Douglas (Thesis advisor) / Chang, Yung (Committee member) / Touchman, Jeffery (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