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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
Description
Amongst the most studied of the social insects, the honey bee has a prominent place due to its economic importance and influence on human societies. Honey bee colonies can have over 50,000 individuals, whose activities are coordinated by chemical signals called pheromones. Because these pheromones are secreted from various exocrine glands, the proper development and function of these glands are vital to colony dynamics. In this thesis, I present a study of the developmental ontogeny of the exocrine glands found in the head of the honey bee. In Chapter 2, I elucidate how the larval salivary gland transitions to an adult salivary gland through apoptosis and cell growth, differentiation and migration. I also explain the development of the hypopharyngeal and the mandibular gland using apoptotic markers and cytoskeletal markers like tubulin and actin. I explain the fundamental developmental plan for the formation of the glands and show that apoptosis plays an important role in the transformation toward an adult gland.
ContributorsNath, Rachna (Author) / Gadau, Juergen (Thesis advisor) / Rawls, Alan (Committee member) / Harrison, Jon (Committee member) / Arizona State University (Publisher)
Created2018
Description
Multicellular organisms use precise gene regulation, executed throughout development, to build and sustain various cell and tissue types. Post-transcriptional gene regulation is essential for metazoan development and acts on mRNA to determine its localization, stability, and translation. MicroRNAs (miRNAs) and RNA binding proteins (RBPs) are the principal effectors of post-transcriptional gene regulation and act by targeting the 3'untranslated regions (3'UTRs) of mRNA. MiRNAs are small non-coding RNAs that have the potential to regulate hundreds to thousands of genes and are dysregulated in many prevalent human diseases such as diabetes, Alzheimer's disease, Duchenne muscular dystrophy, and cancer. However, the precise contribution of miRNAs to the pathology of these diseases is not known.
MiRNA-based gene regulation occurs in a tissue-specific manner and is implemented by an interplay of poorly understood and complex mechanisms, which control both the presence of the miRNAs and their targets. As a consequence, the precise contributions of miRNAs to gene regulation are not well known. The research presented in this thesis systematically explores the targets and effects of miRNA-based gene regulation in cell lines and tissues.
I hypothesize that miRNAs have distinct tissue-specific roles that contribute to the gene expression differences seen across tissues. To address this hypothesis and expand our understanding of miRNA-based gene regulation, 1) I developed the human 3'UTRome v1, a resource for studying post-transcriptional gene regulation. Using this resource, I explored the targets of two cancer-associated miRNAs miR-221 and let-7c. I identified novel targets of both these miRNAs, which present potential mechanisms by which they contribute to cancer. 2) Identified in vivo, tissue-specific targets in the intestine and body muscle of the model organism Caenorhabditis elegans. The results from this study revealed that miRNAs regulate tissue homeostasis, and that alternative polyadenylation and miRNA expression patterns modulate miRNA targeting at the tissue-specific level. 3) Explored the functional relevance of miRNA targeting to tissue-specific gene expression, where I found that miRNAs contribute to the biogenesis of mRNAs, through alternative splicing, by regulating tissue-specific expression of splicing factors. These results expand our understanding of the mechanisms that guide miRNA targeting and its effects on tissue-specific gene expression.
MiRNA-based gene regulation occurs in a tissue-specific manner and is implemented by an interplay of poorly understood and complex mechanisms, which control both the presence of the miRNAs and their targets. As a consequence, the precise contributions of miRNAs to gene regulation are not well known. The research presented in this thesis systematically explores the targets and effects of miRNA-based gene regulation in cell lines and tissues.
I hypothesize that miRNAs have distinct tissue-specific roles that contribute to the gene expression differences seen across tissues. To address this hypothesis and expand our understanding of miRNA-based gene regulation, 1) I developed the human 3'UTRome v1, a resource for studying post-transcriptional gene regulation. Using this resource, I explored the targets of two cancer-associated miRNAs miR-221 and let-7c. I identified novel targets of both these miRNAs, which present potential mechanisms by which they contribute to cancer. 2) Identified in vivo, tissue-specific targets in the intestine and body muscle of the model organism Caenorhabditis elegans. The results from this study revealed that miRNAs regulate tissue homeostasis, and that alternative polyadenylation and miRNA expression patterns modulate miRNA targeting at the tissue-specific level. 3) Explored the functional relevance of miRNA targeting to tissue-specific gene expression, where I found that miRNAs contribute to the biogenesis of mRNAs, through alternative splicing, by regulating tissue-specific expression of splicing factors. These results expand our understanding of the mechanisms that guide miRNA targeting and its effects on tissue-specific gene expression.
ContributorsKotagama, Kasuen Indrajith Bandara (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Joshua (Committee member) / Newbern, Jason (Committee member) / Rawls, Alan (Committee member) / Arizona State University (Publisher)
Created2019
Description
Multiple myeloma is a genetically heterogeneous disease, which can be divided into several genetic subtypes based upon gene expression profiles and chromosomal abnormalities. Unlike older techniques employed in myeloma research, such as cytogenetics, FISH, and microarray technologies, RNA sequencing offers a unique approach to examine the aforementioned genetic characteristics in that it allows for gene expression profiling and the detection of novel fusion transcripts arising from chromosomal rearrangements. This study utilized RNA sequencing to analyze the transcriptomes of 84 multiple myeloma patients and 69 human myeloma cell lines. FCHSD2 was found to be involved in five novel fusion events along with known oncogenes, MMSET and MYC, as well as three previously unreported genes in myeloma, including CHMP4B, NCF2, and CARNS1. An analysis of FCHSD2 expression within myeloma cell lines indicated that it is highly expressed in comparison to other tissues, suggesting that FCHSD2 translocations could lead to promoter replacement events in which the expression of partnering genes is dysregulated. The presence of the five FCHSD2 hybrid transcripts was confirmed by reverse transcription-PCR and Sanger sequencing. Overexpression of the FCHSD2 fusion transcripts in HEK293 cells resulted in the production of N-terminally truncated fusion partner proteins and a novel FCHSD2-CARNS1 fusion protein.
ContributorsMurray, Christopher William (Author) / Wilson-Rawls, Jeanne (Thesis director) / Carpten, John (Committee member) / Keats, Jonathan (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
While a number of vertebrates, including fishes, salamanders, frogs, and lizards, display regenerative capacity, the process is not necessarily the same. It has been proposed that regeneration, while evolutionarily conserved, has diverged during evolution. However, the extent to which the mechanisms of regeneration have changed between taxa still remains elusive. In the salamander limb, cells dedifferentiate to a more plastic state and aggregate in the distal portion of the appendage to form a blastema, which is responsible for outgrowth and tissue development. In contrast, no such mechanism has been identified in lizards, and it is unclear to what extent evolutionary divergence between amniotes and anamniotes has altered this mechanism. Anolis carolinensis lizards are capable of regenerating their tails after stress-induced autotomy or self-amputation. In this investigation, the distribution of proliferating cells in early A. carolinensis tail regeneration was visualized by immunohistochemistry to examine the location and quantity of proliferating cells. An aggregate of proliferating cells at the distal region of the regenerate is considered indicative of blastema formation. Proliferating cell nuclear antigen (PCNA) and minichromosome maintenance complex component 2 (MCM2) were utilized as proliferation markers. Positive cells were counted for each tail (n=9, n=8 respectively). The percent of proliferating cells at the tip and base of the regenerating tail were compared with a one-way ANOVA statistical test. Both markers showed no significant difference (P=0.585, P=0.603 respectively) indicating absence of a blastema-like structure. These results suggest an alternative mechanism of regeneration in lizards and potentially other amniotes.
ContributorsTokuyama, Minami Adrianne (Author) / Kusumi, Kenro (Thesis director) / Wilson-Rawls, Jeanne (Committee member) / Menke, Douglas (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2014-05
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
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
Schizophrenia risk is influenced by both genetic and environmental factors. The immediate early gene early growth response 3 (Egr3), is regulated downstream of several schizophrenia risk genes and encodes a zinc-finger transcription factor protein. Previous studies from our lab indicate that Egr3 deficient (Egr3 -/-) mice exhibit schizophrenia-like phenotypes. We also discovered decreased serotonin 2a receptors (5-HT2AR) in the Egr3 -/- mice, similar to studies that reported decreased 5-HT2ARs in schizophrenia patients. We previously reported that sleep deprivation, a mild stress, causes the over expression of Egr3 and the serotonin 2a gene (Htr2a) in the cortex. To determine whether EGR3, a transcription factor, regulates Htr2a in the prefrontal cortex after sleep deprivation, Egr3 -/-and Egr3 +/+ mice were sleep deprived for eight hours. Transgenic mice were used that expressed enhanced green fluorescent protein (EGFP) under control of the Htr2a promoter via a bacterial artificial chromosome (BAC). Immunohistochemistry was performed to identify EGFP containing cells. Data analysis revealed no significant interaction between genotype and sleep deprivation in 5-HT2AR/EGFP containing cells within the prefrontal cortex. Based on the findings of this study, more data is needed to better determine the relationship between sleep deprivation and its effect on the regulation of Htr2a through in an EGR3 dependent manner.
ContributorsReznik, Derek Lee (Author) / Wilson-Rawls, Jeanne (Thesis director) / Gallitano, Amelia (Committee member) / Anderson, Karen (Committee member) / School of Sustainability (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Adrenocortical carcinoma (ACC) is a rare and deadly disease that affects 0.5-2 people per million per year in the US. Currently, the first line clinical management includes surgical resection, followed by treatment with the chemotherapeutic agent mitotane. These interventions, however, have limited effectiveness, as the overall five-year survival rate of patients with ACC is less than 35%. Therefore, further scientific investigation underlying the molecular mechanisms and biomarkers of this disease is of high importance. The aim of this project was to identify potential biomarkers that may be used as prognosticators as well as candidate genes that might be targeted to develop new therapies for patients with ACC. An analysis of publicly-available datasets revealed PDZ-binding kinase (PBK) as being upregulated roughly 9-fold in ACC tissue compared to normal adrenal tissue. PBK has been implicated as an oncogene in several other systems, and its expression has been shown to negatively impact patient survival. Initial experiments have confirmed the upregulation of PBK in H295R cells, a human ACC cell line. We effectively silenced PBK (>95% reduction in protein content) in H295R cells using lentiviral shRNA constructs. Using high and low PBK expressing cells, we performed soft agar assays for colony formation, and found that the PBK-silenced cells produced two-fold fewer colonies than the vector control (p<0.05). This indicates that PBK likely plays a role in tumorigenicity. We further conducted functional studies for apoptosis and proliferation to elucidate the mechanism by which PBK increases tumorigenicity. Preliminary results from MTS assays showed that after 9 days, PBK-silenced cells proliferated significantly less than the vector control, so PBK likely increases proliferation. Together these data identify PBK as a kinase implicated in ACC tumorigenesis. Further in vitro and in vivo studies will be conducted to evaluate PBK as a potential therapeutic target in adrenocortical carcinoma.
ContributorsRazzaghi, Raud (Author) / Wilson-Rawls, Jeanne (Thesis director) / Anderson, Karen (Committee member) / Katja, Kiseljak-Vassiliades (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12