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- Creators: Marine Biological Laboratory Archives
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
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
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
A literature review summarizing the current status of conservation efforts of the Mojave Desert tortoise (Gopherus agassizii) including a brief overview of the Endangered Species Act (ESA) and its applicability to this species' conservation. A genetic and physiological comparison of the morphologically similar Mojave species with the Sonoran (Gopherus morafkai) species proceeded by an analysis of if and how the ESA should apply to the Sonoran population. Analysis of current plans and interagency cooperations followed by a multi-step proposal on how best to conserve the Sonoran population of Desert tortoise.
ContributorsKulik, Elise Chikako (Author) / Kusumi, Kenro (Thesis director) / Tollis, Marc (Committee member) / Wilson Sayres, Melissa (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
The modern tetraploid species Gossypium barbadense L. (AD2) traces its origins to an allopolyploidy event between diploid progenitors G. raimondii (DT Genome, Americas) and G. herbaceum (AT Genome, Asia/Africa). In this study, nine fiber-related genes consisting of seven MYB transcription factors, a cellulose synthase homolog, and a tubulin homolog were resequenced across 54 G. barbadense lines spanning the wild-to-domesticated spectrum. Tests for nucleotide diversity (π), linkage disequilibrium (LD), and Tajima’s D were performed to examine the extent to which evolutionary forces have acted on these nine loci in G. barbadense. Results indicated that the AT-genome loci had significantly higher levels of diversity and lower levels of LD relative to homoelogous loci from the DT-genome. Additionally, all loci showed signatures of a population size expansion after a bottleneck or selective sweep and/or purifying selection. As previously shown for a sister tetraploid taxa (G. hirsutum), gene conversion resulting from a DT-genome allele invasion into the AT-genome likely explains the higher levels of diversity and lower levels of intragenic LD in the AT-genome. Given the relatively very low level of genetic diversity in elite lines, introduction of novel alleles from wild, land race, or obsolete lines into modern Pima cotton breeding programs is needed to expand the narrow gene pool of G. barbadense for continual yield improvements.
ContributorsNadon, Brian Davis (Author) / Gaxiola, Roberto (Thesis director) / Kusumi, Kenro (Committee member) / Dyer, John (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
The ringtail (Bassariscus astutus), a member of the Procyonidae, is capable of 180 degrees of hindlimb reversal during headfirst descent down a vertical substrate. The goal of this study was to determine the presence or absence of myological adaptations related to hindlimb reversal in the ringtail. Data for B. astutus are presented, including muscle weights and muscle maps ascertained from the dissection of four hindlimbs. Data from the current study were compared to published accounts of other species capable of hindlimb reversal, including procyonids (raccoon, coati, kinkajou, olingo), a mustelid (marten), palm civet, mongoose, tree squirrel, common tree shrew, and slow loris. Muscle mass data from this study demonstrate that the hip adductors of scansorial mammals are significantly more robust than those of terrestrial mammals, indicating a myological adaptation for climbing, but not necessarily hindlimb reversal. Among hindlimb reversers, the majority exhibit one belly of m. sartorius, the presence of m. extensor digiti I longus, and a fibular origin for m. fibularis longus. These characteristics indicate an emphasis on hip extension, ankle plantarflexion, and pes inversion. However, these characteristics are more likely due to phylogeny than hindlimb reversal because of their presence in closely-related non-reversers. Additional data on families outside of Carnivora may help determine if these myological traits are indeed due to phylogeny. Other myological data, such as moment arms and cross sectional areas, may provide evidence of adaptations for hindlimb reversal.
ContributorsLiu, Margaret Chuan (Author) / Fisher, Rebecca (Thesis director) / Hinrichs, Richard (Committee member) / Kusumi, Kenro (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05