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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
The use of synthetic cathinones or "bath salts" has risen dramatically in recent years with one of the most popular being Methylendioxypyrovalerone (MDPV). Following the temporary legislative ban on the sale and distribution of this compound , a multitude of other cathinone derivatives have been synthesized. The current study seeks to compare the abuse potential of MDPV with one of the emergent synthetic cathinones 4-methylethcathinone (4-MEC), based on their respective ability to lower current thresholds in an intracranial self-stimulation (ICSS) paradigm. Following acute administration (0.1, 0.5, 1 and 2 mg/kg i.p.) MDPV was found to significantly lower ICSS thresholds at all doses tested (F4,35=11.549, p<0.001). However, following acute administration (0.3,1,3,10,30 mg/kg i.p) 4-MEC produced no significant ICSS threshold depression (F5,135= 0.622, p = 0.684). Together these findings suggest that while MDPV may possess significant abuse potential, other synthetic cathinones such as 4-MEC may have a drastically reduced potential for abuse.
ContributorsWegner, Scott Andrew (Author) / Olive, M. Foster (Thesis director) / Presson, Clark (Committee member) / Sanabria, Federico (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Department of Psychology (Contributor)
Created2013-05
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
Description
Skeletal muscles arise from the myotome compartment of the somites that form during vertebrate embryonic development. Somites are transient structures serve as the anlagen for the axial skeleton, skeletal muscle, tendons, and dermis, as well as imposing the metameric patterning of the axial musculoskeletal system, peripheral nerves, and vasculature. Classic studies have described the role of Notch, Wnt, and FGF signaling pathways in controlling somite formation and muscle formation. However, little is known about the transformation of myotome compartments into identifiable post-natal muscle groups. Using a mouse model, I have undertaken an evaluation of morphological events, including hypertrophy and hyperplasia, related to the formation of several muscles positioned along the dorsal surface of the vertebrae and ribs. Lunatic fringe (Lfng) deficient embryos and neonates were also examined to further understand the role of the Notch pathway in these processes as it is a modulator of the Notch receptor and plays an important role in defining somite borders and anterior-posterior patterning in many vertebrates. Lunatic fringe deficient embryos showed defects in muscle fiber hyperplasia and hypertrophy in the iliocostalis and longissimus muscles of the erector spinae group. This novel data suggests an additional role for Lfng and the Notch signaling pathway in embryonic and fetal muscle development.
ContributorsDe Ruiter, Corinne (Author) / Rawls, J. Alan (Thesis advisor) / Wilson-Rawls, Jeanne (Committee member) / Kusumi, Kenro (Committee member) / Fisher, Rebecca E. (Committee member) / Arizona State University (Publisher)
Created2012
Description
Postnatal skeletal muscle repair is dependent on the tight regulation of an adult stem cell population known as satellite cells. In response to injury, these quiescent cells are activated, proliferate and express skeletal muscle-specific genes. The majority of satellite cells will fuse to damaged fibers or form new muscle fibers, while a subset will return to a quiescent state, where they are available for future rounds of repair. Robust muscle repair is dependent on the signals that regulate the mutually exclusive decisions of differentiation and self-renewal. A likely candidate for regulating this process is NUMB, an inhibitor of Notch signaling pathway that has been shown to asymmetrically localize in daughter cells undergoing cell fate decisions. In order to study the role of this protein in muscle repair, an inducible knockout of Numb was made in mice. Numb deficient muscle had a defective repair response to acute induced damage as characterized by smaller myofibers, increased collagen deposition and infiltration of fibrotic cells. Satellite cells isolated from Numb-deficient mice show decreased proliferation rates. Subsequent analyses of gene expression demonstrated that these cells had an aberrantly up-regulated Myostatin (Mstn), an inhibitor of myoblast proliferation. Further, this defect could be rescued with Mstn specific siRNAs. These data indicate that NUMB is necessary for postnatal muscle repair and early proliferative expansion of satellite cells. We used an evolutionary compatible to examine processes controlling satellite cell fate decisions, primary satellite cell lines were generated from Anolis carolinensis. This green anole lizard is evolutionarily the closet animal to mammals that forms de novo muscle tissue while undergoing tail regeneration. The mechanism of regeneration in anoles and the sources of stem cells for skeletal muscle, cartilage and nerves are poorly understood. Thus, satellite cells were isolated from A. carolinensis and analyzed for their plasticity. Anole satellite cells show increased plasticity as compared to mouse as determined by expression of key markers specific for bone and cartilage without administration of exogenous morphogens. These novel data suggest that satellite cells might contribute to more than muscle in tail regeneration of A. carolinensis.
ContributorsGeorge, Rajani M (Author) / Wilson-Rawls, Jeanne (Thesis advisor) / Rawls, Alan (Committee member) / Whitfield, Kerr (Committee member) / Kusumi, Kenro (Committee member) / Arizona State University (Publisher)
Created2012
Description
Background: Both puberty and diets composed of high levels of saturated fats have been shown to result in central adiposity, fasting hyperinsulinemia, insulin resistance and impaired glucose tolerance. While a significantly insulinogenic phenotypic change occurs in these two incidences, glucose homeostasis does not appear to be affected. Methods: Male, Sprague-dawley rats were fed diets consisting of CHOW or low fat (LF), High Fat Diet and High Fat Diet (HFD) with supplementary Canola Oil (Monounsaturated fat). These rats were given these diets at 4-5 weeks old and given intraperitoneal and oral glucose tolerance tests(IPGTT; OGTT) at 4 and 8 weeks to further understand glucose and insulin behavior under different treatments. (IPGTT: LF-n=14, HFD-n=16, HFD+CAN-n=12; OGTT: LF-n=8, HFD-n=8, HFD+CAN-n=6). Results: When comparing LF fed rats at 8 weeks with 4 week glucose challenge test, area under the curve (AUC) of glucose was 1.2 that of 4 weeks. At 8 weeks, HFD fed rats AUCg was much greater than LF fed rats under both IPGTT and OGTT. When supplemented with Canola oil, HFD fed rats AUC returned to LF data range. Despite the alleviating glucose homeostasis affects of Canola oil the AUC of insulin curve, which was elevated by HFD, remained high. Conclusion: HFD in maturing rats elevates fasting insulin levels, increases insulin resistance and lowers glucose homeostasis. When given a monounsaturated fatty acid (MUFA) supplement fasting hyperinsulinemia, and late hyperinsulinemia still occur though glucose homeostasis is regained. For OGTT HFD also induced late hyper c-peptide levels and compared to LF and HFD+CAN, a higher c-peptide level over time.
ContributorsRay, Tyler John (Author) / Caplan, Michael (Thesis director) / Herman, Richard (Committee member) / Towner, Kali (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / W. P. Carey School of Business (Contributor) / School of Human Evolution and Social Change (Contributor)
Created2015-05
Description
Nucleic acids encode the information required to create life, and polymerases are the gatekeepers charged with maintaining the storage and flow of this genetic information. Synthetic biologists utilize this universal property to modify organisms and other systems to create unique traits or improve the function of others. One of the many realms in synthetic biology involves the study of biopolymers that do not exist naturally, which is known as xenobiology. Although life depends on two biopolymers for genetic storage, it may be possible that alternative molecules (xenonucleic acids – XNAs), could be used in their place in either a living or non-living system. However, implementation of an XNA based system requires the development of polymerases that can encode and decode information stored in these artificial polymers. A strategy called directed evolution is used to modify or alter the function of a protein of interest, but identifying mutations that can modify polymerase function is made problematic by their size and overall complexity. To reduce the amount of sequence space that needs to be samples when attempting to identify polymerase variants, we can try to make informed decisions about which amino acid residues may have functional roles in catalysis. An analysis of Family B polymerases has shown that residues which are involved in substrate specificity are often highly conserved both at the sequence and structure level. In order to validate the hypothesis that a strong correlation exists between structural conservation and catalytic activity, we have selected and mutated residues in the 9°N polymerase using a loss of function mutagenesis strategy based on a computational analysis of several homologues from a diverse range of taxa. Improvement of these models will hopefully lead to quicker identification of loci which are ideal engineering targets.
ContributorsHaeberle, Tyler Matthew (Author) / Chaput, John (Thesis director) / Chen, Julian (Committee member) / Larsen, Andrew (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Lung cancer is the leading cause of cancer-related deaths in the US. Low-dose computed tomography (LDCT) scans are speculated to reduce lung cancer mortality. However LDCT scans impose multiple risks including false-negative results, false- positive results, overdiagnosis, and cancer due to repeated exposure to radiation. Immunosignaturing is a new method proposed to screen and detect lung cancer, eliminating the risks associated with LDCT scans. Known and blinded primary blood sera from participants with lung cancer and no cancer were run on peptide microarrays and analyzed. Immunosignatures for each known sample collectively indicated 120 peptides unique to lung cancer and non-cancer participants. These 120 peptides were used to determine the status of the blinded samples. Verification of the results from Vanderbilt is pending.
ContributorsNguyen, Geneva Trieu (Author) / Woodbury, Neal (Thesis director) / Zhao, Zhan-Gong (Committee member) / Stafford, Phillip (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Department of Psychology (Contributor)
Created2015-05