Matching Items (52)
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
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
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
Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) is a rare and highly aggressive ovarian cancer that affects children and young women at a mean age of 24 years. Most SCCOHT patients are diagnosed at an advanced stage and do not respond to chemotherapy. As a result, more than 75% of patients succumb to their disease within 1-2 years. To provide insights into the biological, diagnostic, and therapeutic vulnerabilities of this deadly cancer, a comprehensive characterization of 22 SCCOHT cases and 2 SCCOHT cell lines using microarray and next-generation sequencing technologies was performed. Following histological examination, tumor DNA and RNA were extracted and used for array comparative genomic hybridization and gene expression microarray analyses. In agreement with previous reports, SCCOHT presented consistently diploid profiles with few copy number aberrations. Gene expression analysis showed SCCOHT tumors have a unique gene expression profile unlike that of most common epithelial ovarian carcinomas. Dysregulated cell cycle control, DNA repair, DNA damage-response, nucleosome assembly, neurogenesis and nervous system development were all characteristic of SCCOHT tumors. Sequencing of DNA from SCCOHT patients and cell lines revealed germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 79% (19/24) of SCCOHT patients in addition to SMARCA4 protein loss in 84% (16/19) of SCCOHT tumors, but in only 0.4% (2/485) of other primary ovarian tumors. Ongoing studies are now focusing on identifying treatments for SCCOHT based on therapeutic vulnerabilities conferred by ubiquitous inactivating mutations in SMARCA4 in addition to gene and protein expression data. Our characterization of the molecular landscape of SCCOHT and the breakthrough identification of inactivating SMARCA4 mutations in almost all cases of SCCOHT offers the first significant insight into the molecular pathogenesis of this disease. The loss of SMARCA4 protein is a highly sensitive and specific marker of the disease, highlighting its potential role as a diagnostic marker, and offers the opportunity for genetic testing of family members at risk. Outstanding questions remain about the role of SMARCA4 loss in the biology, histogenesis, diagnosis, and treatment of SCCOHT.
ContributorsRamos, Pilar (Author) / Anderson, Karen (Thesis advisor) / Trent, Jeffrey (Committee member) / Kusumi, Kenro (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2014
Description
During the downswing all golfers must roll their forearms and twist the club handle in order to square the club face into impact. Anecdotally some instructors say that rapidly twisting the handle and quickly closing the club face is the best technique while others disagree and suggest the opposite. World class golfers have swings with a range of club handle twist velocities (HTV) from very slow to very fast and either method appears to create a successful swing. The purpose of this research was to discover the relationship between HTV at impact and selected body and club biomechanical characteristics during a driver swing. Three-dimensional motion analysis methods were used to capture the swings of 94 tour professionals. Pearson product-moment correlation was used to determine if a correlation existed between HTV and selected biomechanical characteristics. The total group was also divided into two sub-groups of 32, one group with the fastest HTV (Hi-HTV) and the other with the slowest HTV (Lo-HTV). Single factor ANOVAs were completed for HTV and each selected biomechanical parameter. No significant differences were found between the Hi-HTV and Lo-HTV groups for both clubhead speed and driving accuracy. Lead forearm supination velocity at impact was found to be significantly different between groups with the Hi-HTV group having a higher velocity. Lead wrist extension velocity at impact, while not being significantly different between groups was found to be positive in both groups, meaning that the lead wrist is extending at impact. Lead wrist ulnar deviation, lead wrist release and trail elbow extension velocities at maximum were not significantly different between groups. Pelvis rotation, thorax rotation, pelvis side bend and pelvis rotation at impact were all significantly different between groups, with the Lo-HTV group being more side bent tor the trail side and more open at impact. These results suggest that world class golfers can successfully use either the low or high HTV technique for a successful swing. From an instructional perspective it is important to be aware of the body posture and wrist/forearm motion differences between the two techniques so as to be consistent when teaching either method.
ContributorsCheetham, Phillip (Author) / Hinrichs, Richard (Thesis advisor) / Ringenbach, Shannon (Committee member) / Dounskaia, Natalia (Committee member) / Crews, Debra (Committee member) / Arizona State University (Publisher)
Created2014
Description
Skeletal muscle injury may occur from repetitive short bursts of biomechanical strain that impair muscle function. Alternatively, variations of biomechanical strain such as those held for long-duration are used by clinicians to repair muscle and restore its function. Fibroblasts embedded within the unifying connective tissue of skeletal muscle experience these multiple and diverse mechanical stimuli and respond by secreting cytokines. Cytokines direct all stages of muscle regeneration including myoblasts differentiation, fusion to form myotubes, and myotube functionality. To examine how fibroblasts respond to variations in mechanical strain that may affect juxtapose muscle, a myofascial junction was bioengineered that examined the interaction between the two cell types. Fibroblasts were experimentally shown to increase myoblast differentiation, and fibroblast biomechanical strain mediated the extent to which differentiation occurred. Intereleukin-6 is a strain-regulated cytokine secreted by fibroblasts was determined to be necessary for fibroblast-mediated myoblast differentiation. Myotubes differentiated in the presence of strained fibroblasts express greater number of acetylcholine receptors, greater acetylcholine receptor sizes, and modified to be more or less sensitive to acetylcholine-induced contraction. This study provides direct evidence that strained and non-strained fibroblasts can serve as a vehicle to modify myoblast differentiation and myotube functionality. Further understanding the mechanisms regulating these processes may lead to clinical interventions that include strain-activated cellular therapies and bioengineered cell engraftment for mediating the regeneration and function of muscle in vivo.
ContributorsHicks, Michael (Author) / Standley, Paul R (Thesis advisor) / Rawls, Jeffrey (Committee member) / Lake, Douglas (Committee member) / Hinrichs, Richard (Committee member) / Arizona State University (Publisher)
Created2014
Description
Telomerase enzyme is a truly remarkable enzyme specialized for the addition of short, highly repetitive DNA sequences onto linear eukaryotic chromosome ends. The telomerase enzyme functions as a ribonucleoprotein, minimally composed of the highly conserved catalytic telomerase reverse transcriptase and essential telomerase RNA component containing an internalized short template region within the vastly larger non-coding RNA. Even among closely related groups of species, telomerase RNA is astonishingly divergent in sequence, length, and secondary structure. This massive disparity is highly prohibitive for telomerase RNA identification from previously unexplored groups of species, which is fundamental for secondary structure determination. Combined biochemical enrichment and computational screening methods were employed for the discovery of numerous telomerase RNAs from the poorly characterized echinoderm lineage. This resulted in the revelation that--while closely related to the vertebrate lineage and grossly resembling vertebrate telomerase RNA--the echinoderm telomerase RNA central domain varies extensively in structure and sequence, diverging even within echinoderms amongst sea urchins and brittle stars. Furthermore, the origins of telomerase RNA within the eukaryotic lineage have remained a persistent mystery. The ancient Trypanosoma telomerase RNA was previously identified, however, a functionally verified secondary structure remained elusive. Synthetic Trypanosoma telomerase was generated for molecular dissection of Trypanosoma telomerase RNA revealing two RNA domains functionally equivalent to those found in known telomerase RNAs, yet structurally distinct. This work demonstrates that telomerase RNA is uncommonly divergent in gross architecture, while retaining critical universal elements.
ContributorsPodlevsky, Joshua (Author) / Chen, Julian (Thesis advisor) / Mangone, Marco (Committee member) / Kusumi, Kenro (Committee member) / Wilson-Rawls, Norma (Committee member) / Arizona State University (Publisher)
Created2015
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
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
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