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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
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
The development of skeletal muscle during embryogenesis and repair in adults is dependent on the intricate balance between the proliferation of myogenic progenitor cells and the differentiation of those cells into functional muscle fibers. Recent studies demonstrate that the Drosophila melanogaster transcription factor CG9650 is expressed in muscle progenitor cells, where it maintains myoblast numbers. We are interested in the Mus musculus orthologs Bcl11a and Bcl11b (C2H2 zinc finger transcription factors), and understanding their role as molecular switches that control proliferation/differentiation decisions in muscle progenitor cells. Expression analysis revealed that Bcl11b, but not Bcl11a, is expressed in the region of the mouse embryo populated with myogenic progenitor cells; gene expression studies in muscle cell culture confirmed Bcl11b is also selectively transcribed in muscle. Furthermore, Bcl11b is down-regulated with differentiation, which is consistent with the belief that the gene plays a role in cell proliferation.
ContributorsDuong, Brittany Bach (Author) / Rawls, Alan (Thesis director) / Wilson-Rawls, Jeanne (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
Background: Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer deaths in females worldwide, accounting for 23% of all new cancer cases and 14% of all total cancer deaths in 2008. Five tumor-normal pairs of primary breast epithelial cells were treated for infinite proliferation by using a ROCK inhibitor and mouse feeder cells. Methods: Raw paired-end, 100x coverage RNA-Seq data was aligned to the Human Reference Genome Version 19 using BWA and Tophat. Gene differential expression analysis was completed using Cufflinks and Cuffdiff. Interactive Genome Viewer was used for data visualization. Results: 15 genes were found to be down-regulated by at least one log-fold change in 4/5 of tumor samples. 75 genes were found to be down-regulated in 3/5 of our tumor samples by at least one log-fold change. 11 genes were found to be up-regulated in 4/5 of our tumor samples, and 68 genes were identified to be up-regulated in 3/5 of the tumor samples by at least one-fold change. Conclusion: Expression changes in genes such as AZGP1, AGER, ALG11, and S1007 suggest a disruption in the glycosylation pathway. No correlation was found between Cufflink's Her2 gene-expression and DAKO score classification.
ContributorsHernandez, Fernando (Author) / Anderson, Karen (Thesis director) / Mangone, Marco (Committee member) / Park, Jin (Committee member) / Barrett, The Honors College (Contributor) / Department of Information Systems (Contributor)
Created2013-05
Description
Five immunocompetent C57BL/6-cBrd/cBrd/Cr (albino C57BL/6) mice were injected with GL261-luc2 cells, a cell line sharing characteristics of human glioblastoma multiforme (GBM). The mice were imaged using magnetic resonance (MR) at five separate time points to characterize growth and development of the tumor. After 25 days, the final tumor volumes of the mice varied from 12 mm3 to 62 mm3, even though mice were inoculated from the same tumor cell line under carefully controlled conditions. We generated hypotheses to explore large variances in final tumor size and tested them with our simple reaction-diffusion model in both a 3-dimensional (3D) finite difference method and a 2-dimensional (2D) level set method. The parameters obtained from a best-fit procedure, designed to yield simulated tumors as close as possible to the observed ones, vary by an order of magnitude between the three mice analyzed in detail. These differences may reflect morphological and biological variability in tumor growth, as well as errors in the mathematical model, perhaps from an oversimplification of the tumor dynamics or nonidentifiability of parameters. Our results generate parameters that match other experimental in vitro and in vivo measurements. Additionally, we calculate wave speed, which matches with other rat and human measurements.
ContributorsRutter, Erica (Author) / Stepien, Tracy (Author) / Anderies, Barrett (Author) / Plasencia, Jonathan (Author) / Woolf, Eric C. (Author) / Scheck, Adrienne C. (Author) / Turner, Gregory H. (Author) / Liu, Qingwei (Author) / Frakes, David (Author) / Kodibagkar, Vikram (Author) / Kuang, Yang (Author) / Preul, Mark C. (Author) / Kostelich, Eric (Author) / College of Liberal Arts and Sciences (Contributor)
Created2017-05-31
Description
Technologies capable of characterizing the full breadth of cellular systems need to be able to measure millions of proteins, isoforms, and complexes simultaneously. We describe an approach that fulfils this criterion: Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT). ADAPT employs an enriched library of single-stranded oligodeoxynucleotides (ssODNs) to profile complex biological samples, thus achieving an unprecedented coverage of system-wide, native biomolecules. We used ADAPT as a highly specific profiling tool that distinguishes women with or without breast cancer based on circulating exosomes in their blood. To develop ADAPT, we enriched a library of ~10[superscript 11] ssODNs for those associating with exosomes from breast cancer patients or controls. The resulting 10[superscript 6] enriched ssODNs were then profiled against plasma from independent groups of healthy and breast cancer-positive women. ssODN-mediated affinity purification and mass spectrometry identified low-abundance exosome-associated proteins and protein complexes, some with known significance in both normal homeostasis and disease. Sequencing of the recovered ssODNs provided quantitative measures that were used to build highly accurate multi-analyte signatures for patient classification. Probing plasma from 500 subjects with a smaller subset of 2000 resynthesized ssODNs stratified healthy, breast biopsy-negative, and -positive women. An AUC of 0.73 was obtained when comparing healthy donors with biopsy-positive patients.
ContributorsDomenyuk, Valeriy (Author) / Zhong, Zhenyu (Author) / Stark, Adam (Author) / Xiao, Nianqing (Author) / O'Neill, Heather A. (Author) / Wei, Xixi (Author) / Wang, Jie (Author) / Tinder, Teresa T. (Author) / Tonapi, Sonal (Author) / Duncan, Janet (Author) / Hornung, Tassilo (Author) / Hunter, Andrew (Author) / Miglarese, Mark R. (Author) / Schorr, Joachim (Author) / Halbert, David D. (Author) / Quackenbush, John (Author) / Poste, George (Author) / Berry, Donald A. (Author) / Mayer, Gunter (Author) / Famulok, Michael (Author) / Spetzler, David (Author) / Consortium for Biosocial Complex Systems (Contributor) / Complex Adaptive Systems Initiative (Contributor)
Created2017-02-20
Description
The Numb gene encodes an adaptor protein that has been shown to play a role in muscle repair, cell proliferation, and the determination of cell fate in satellite cells. Ablation of this gene in satellite cells results in an up-regulation of myostatin and p21, which inhibit the proliferation of myoblasts. These results indicate that the regulation of numb and myostatin could be used to amplify muscle regeneration. This would function as a therapeutic approach to degenerative muscle diseases, such as muscular dystrophy. There are four mammalian NUMB proteins produced through alternative splicing of the Numb mRNA transcript. Only two isoforms are present in adult mammalian muscle, indicating some form of muscle-specific post-transcriptional control of the gene. Additionally, the presence of two polyadenylation sites, and multiple miRNA seed sequences within the 3’ untranslated region (UTR) of mouse Numb indicate the possibility of regulation by a muscle specific miRNA.
ContributorsGefroh, Bailey Emelia (Co-author) / Gefroh, Bailey (Co-author) / Wilson-Rawls, Jeanne (Thesis director) / Rawls, Alan (Committee member) / Palade, Joanna (Committee member) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-12
Description
Sarcopenia, a disease defined by age-related muscle loss and function, impacts each and every one of us as we age. Medical research over the past 40 years has identified dozens of factors that contribute to Sarcopenia, including, hormonal changes, deficiencies in nutrition, denervation, changes in physical activity and diseases. Developing effective therapeutic treatments for Sarcopenia is dependent on identifying the mechanisms by which these factors affect muscle loss and understanding the interrelationship of these mechanisms. I conducted my research by compiling and analyzing several previous studies on many different mechanisms that contribute to Sarcopenia. Of these mechanisms, I determined the most significant mechanisms and mapped them out on a visual presentation. In addition to the contributing factors listed above, I found that dysregulated cell signaling, mitochondrial abnormalities, impaired autophagy/protein regulation, altered nitric oxide production, and systemic inflammation all contribute to Sarcopenia. Their impact on skeletal muscle is manifested by reduced satellite function, reduced regenerative capacity, loss of muscle mass, accumulation of damaged products, and fibrosis. My research clearly demonstrated that there was not a one-to-one correlation between factors and specific pathological characteristics of Sarcopenia. Instead, factors funneled into a discrete number of cellular processes, including cell proliferation, protein synthesis, and autophagy and apoptosis. Based on my findings, the overall cause of Sarcopenia appears to be a loss of balance between these pathways. The results of my thesis indicate that Sarcopenia is a multifactorial disorder, and therefore, effective therapy should consist of those that prevent necrosis associated with autophagy and apoptosis.
ContributorsSmith, Cameron Isaiah (Co-author) / Rawls, Alan (Co-author, Thesis director) / Wilson-Rawls, Jeanne (Committee member) / School of Life Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Numb is a gene that encodes an adaptor protein which has been characterized for its role cell migration, cell adhesion, endocytosis, and cell fate determination through asymmetrical division in various embryonic and adult tissues. In vertebrates, several Numb isoforms are produced via alternative splicing. In the Mus musculus genome, one Numb gene on chromosome 12 is alternatively spliced to produce four distinct protein isoforms, characterized by an 11 amino acid insert in the phosphotyrosine binding domain and a 49 amino acid insert in the proline rich region. Two poly adenylation sites in the currently published Numb 3' UTR exist, thus, the possibility that various 3' UTRs containing different miRNA seed sites is a possible posttranscriptional mechanism by which differential expression is observed. In an attempt to elucidate this hypothesis, PCR was performed to amplify the 3' UTR of murine neural tube cells, the products of which were subsequently cloned and sequenced. Multiple fragment sizes were consistently observed in the PCR data, however, sequencing demonstrated that these bands did not reveal an association with Numb.
ContributorsGama, Garrick Joseph (Author) / Wilson-Rawls, Jeanne (Thesis director) / Rawls, Alan (Committee member) / Palade, Joanna (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Duchenne Muscular Dystrophy (DMD) is a muscular degenerative disease characterized by striated membrane instability that stimulates continuous cycles of muscle repair. Chronic activation of the innate immune response necessary for muscle repair leads to a pathological accumulation of fibrotic materials that disrupt muscle function. During healthy tissue repair, a balance between pro-inflammatory macrophage (M1) and anti-inflammatory macrophage (M2) promotes clearance of necrotic fibers (myolysis) followed by tissue repair. This is regulated by an intricate feedback loop between muscle, neutrophils and macrophages mediated by Th1 and Th2 cytokines and chemokines. During chronic inflammation, there is an imbalance in an M2 species that produces high levels of extracellular matrix that leads to fibrosis. Finding treatments that ameliorate fibrosis are essential to limiting the muscle pathology that reduces ambulation of DMD patients. Previous studies have shown that Mohawk, (Mkx) a homeobox transcription factor, is essential for the initiation of the inflammation response during acute muscle injury. This study aims to examine whether Mkx regulates inflammation during chronic damage associated with muscular dystrophy. The mdx mouse is a well-studied mouse model that recapitulates muscle necrosis, chronic inflammatory response and fibrosis associated with muscular dystrophy. Utilizing quantitative RT-PCR and histological analysis, the diaphragms and Quadriceps of adult Mkx-/-/mdx and Mkx+/+/mdx mice were analyzed at three critical time points (4 weeks, 3 months and 7 months). In contrast to what was anticipated, there was evidence of increased muscle damage in the absence of Mkx. There was a consistent reduction in the diameter of muscle fibers found in both types of tissue in Mkx-/-/mdx versus Mkx+/+/mdx mice without a difference in the number of fibers with centralized nuclei at 4 weeks and 1 year between the two genotypes, suggesting that the Mkx mutation influences the maturation of fibers forming in response to muscle damage. Fibrosis was higher in the diaphragm of the Mkx-/-/mdx mice at 4 weeks and 3 months, while at1 year there did not appear to be a difference. Overall, the results predict that the absence of Mkx exacerbates the instability of muscle fibers in the mdx mouse. Future studies will be needed to understand the relationship between Mkx and the dystrophin gene.
ContributorsMasson, Samantha Ashley (Author) / Rawls, Alan (Thesis director) / Wilson-Rawls, Jeanne (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05