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- Member of: Barrett, The Honors College Thesis/Creative Project Collection
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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
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
Description
Staufen is a double-stranded RNA binding protein (dsRBP) with discovered homologs in a diverse range of animals, insects, and other multicellular organisms. Staufen acts on secondary structures in mRNA transcripts to modulate translation of many targets through several mechanisms of action. It has roles in microtubule-dependent subcellular localization of mRNA transcripts, translational activation, transcript stability, Staufen-mediated mRNA decay (SMD), is a known component of RNA granules, and has been implicated in several cellular processes, one being myogenesis. Mammals have two Staufen orthologs–Staufen1 and Staufen2. Staufen1 has four conserved dsRNA binding domains (dsRBDs), each with distinct functional characteristics. This study finds that cultured MuSCs show distinct patterns of Staufen1 transcriptional expression from quiescence throughout the myogenic differentiation program characterized by high expression in quiescent satellite cells, less expression in proliferating myoblasts, and fairly high, sustained expression throughout differentiation and myotube formation. The temporal expression pattern is compared with recently reported novel Staufen1 functions in myogenesis. This research highlights that Staufen1 is able to act on transcripts in several overlapping ways to assist in the regulation of myogenesis, and more extensive characterization of Staufen1 as well as high-confidence identification of Staufen binding sites (SBS), will be necessary to fit Staufen1 into a model of translational regulation in myogenesis.
ContributorsLiakos, Nicholas (Author) / Wilson-Rawls, Jeanne (Thesis director) / Diviak, Bridget (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2023-05
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
The highly conserved Notch signaling pathway regulates cell-cell communication pathways, cell fate, cell determination, cell death, embryonic development, and adult tissue pathways in metazoans. The Notch receptors and ligands that bind to Notch are single pass, transmembrane proteins that communicate cell to cell via juxtacrine signaling. There are reports of the divergent function and localization of the Deltalike 3 (Dll3) ligand. In Mus musculus (an eutherin mammal) the DLL3 protein inhibits the Notch signaling pathway and is localized in the Golgi apparatus. In contrast, the DLL3 protein from zebrafish, Danio rerio (a teleost) activates Notch and is located on the cell surface. This study will focus on examining the evolutionary pathway/evolutionary similarities, localization, and function of the A. carolinensis dll3 gene in comparison to other vertebrate species. This is important because there is not much known about the evolutionary divergence of the DLL3 A. carolinensis protein, its function in Notch signaling, and its subcellular localization.
Evolutionary analysis of vertebrate DLL3 protein sequences using phylogenetic trees showed that D. rerio and A. carolinensis are more evolutionarily similar in comparison to M. musculus suggesting that they may have similar intracellular localization. However, immunofluorescence staining experiments showed that the A. carolinensis DLL3 protein co-localized significantly with an endoplasmic reticulum (ER) specific primary antibody. Since this protein is localized in the secretory system, similar to that of M. musculus DLL3, it suggests that its function is to inhibit the Notch signaling pathway. Protein sequence alignments were created that suggested that there is a region in the protein sequences where the lizard and mouse sequence are conserved, while the zebrafish sequence simultaneously varies. This region of the amino acid sequence could be responsible for the difference in localization and function of the protein in these two species.
Evolutionary analysis of vertebrate DLL3 protein sequences using phylogenetic trees showed that D. rerio and A. carolinensis are more evolutionarily similar in comparison to M. musculus suggesting that they may have similar intracellular localization. However, immunofluorescence staining experiments showed that the A. carolinensis DLL3 protein co-localized significantly with an endoplasmic reticulum (ER) specific primary antibody. Since this protein is localized in the secretory system, similar to that of M. musculus DLL3, it suggests that its function is to inhibit the Notch signaling pathway. Protein sequence alignments were created that suggested that there is a region in the protein sequences where the lizard and mouse sequence are conserved, while the zebrafish sequence simultaneously varies. This region of the amino acid sequence could be responsible for the difference in localization and function of the protein in these two species.
ContributorsBoschi, Alexis (Author) / Wilson-Rawls, Jeanne (Thesis director) / Newbern, Jason (Committee member) / Wilson Sayres, Melissa (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05