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- All Subjects: Numb
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
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
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