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Dissertations / Theses on the topic 'Oncogenes – Research'
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1
Scully, Jaqueline Susan. "Insertion of oncogenes into mouse mammary epithelium." Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315287.
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Xie, Dan, and 謝丹. "Application of high-throughput tissue microarray technology in cancer research." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B30283619.
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Dong, Suisui, and 董穗穗. "Applications of T-rex tetracycline inducible expression system on identifying downstream targets of oncogenes in HCC research." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B45456641.
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Schiavi, Susan C. "MYC and E1A Oncogenes Alter the Response of PC12 Cells to Nerve Growth Factor and Block Differentiation: A Thesis." eScholarship@UMMS, 1988. https://escholarship.umassmed.edu/gsbs_diss/259.
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PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) by neuronal differentiation and partial growth arrest. Mouse c-myc and adenovirus E1A genes were introduced into PC12 cells to study the influence of these nuclear oncogenes on neuronal differentiation. Expression of myc and E1A blocked morphological differentiation and caused NGF to stimulate rather than inhibit cell proliferation. NGF binding to cell surface receptors, activation of ribosomal S6 kinase, and ornithine decarboxylase induction were similar in myc and E1A expressing clones compared with wild-type PC12 cells, suggesting that changes in the cellular response to NGF were at a post-receptor level. The ability of myc and E1A expression to block the transcription-dependent induction of microtubule associated proteins by NGF further suggested that these genes may inhibit differentiation by interfering with NGP's ability to regulate transcription. These results illustrate that NGF can promote either growth or differentiation of PC12 cells, and that myc or E1A alter the phenotypic responses to growth factors.
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Appleby, Mark William. "Oncogene expression and the modulation of keratinocyte self renewal." Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.306476.
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Whitelaw, Christopher Bruce Alexander. "An analysis of the transcriptional control domains of the human c-myc proto-oncogene." Thesis, University of Glasgow, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.306235.
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Lucassen, Emy Marian. "The role of the neu oncogene in the transformation and differentiation of mammary epithelial cells." Thesis, Open University, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.276089.
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Thomas, Hilary. "A feasibility study of oncogene transgenic mice as therapeutic models in cytokine research." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264165.
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Sumner, Evan T. "Characterizing the Oncogenic Properties of C-terminal Binding Protein." VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4153.
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The paralogous C-terminal binding proteins (CtBP) 1 and 2 are evolutionarily conserved transcriptional coregulators that target and disrupt the expression of several genes essential for multiple cellular processes critical to regulating tumor formation. CtBP’s ability to govern the transcription of genes necessary for apoptosis, tumor suppression, invasion/migration and EMT gives rise to its oncogenic activities. Both isoforms of CtBP are found to be overexpressed in cancers including colorectal, pancreatic, ovarian, and breast, with higher levels correlating to lower overall median survival. Although multiple lines of evidence suggest CtBP plays a role in tumorigenesis, it has never been formally characterized as an oncogene. For this reason, the goal of this dissertation was to design a set of experiments to determine the transforming ability of CtBP2 in vitro using both murine and human fibroblast and in vivo using the Apcmin/+ mouse model of cancer. Specifically, we demonstrate that overexpression of CtBP2 alone can drive transformation of NIH3T3 cells leading to loss of contact inhibition, increased x invasion/migration, and anchorage independent growth. In addition, CtBP2 was found to cooperate with the large T-antigen (LT) component of the simian virus 40 (SV40) to lead to transformation of murine embryonic fibroblasts (MEFs) and with both LT and small T-antigen (ST) to induce migration/invasion and anchorage-independent growth in BJ human foreskin fibroblasts. To confirm the role of Ctbp2 in a mouse tumor model with Ctbp overexpression, we bred Apcmin/+ mice to Ctbp2 heterozygous (Ctbp2+/-) mice, which otherwise live normal lifespans. CtBP is a known target of the APC tumor suppressor and is thus stabilized in APC mutated human colon cancers and is found in high levels in Apcmin/+ polyps. Remarkably, removing an allele of Ctbp2 doubled the median survival of Apcmin/+ mice (P <0.001) and reduced polyp formation to near undetectable levels. These data suggest the importance of CtBP2 in driving cellular transformation and identify it as a potential target for prevention or therapy in APC mutant backgrounds.
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Knarr, Matthew J. "The Monkey in the Wrench: MiR-181a's Role in Promoting Adipogenesis and Ovarian Cancer Transformation." Case Western Reserve University School of Graduate Studies / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=case1554481048956007.
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Johnson, Kirsten M. "Characterization of length-dependent GGAA-microsatellites in EWS/FLI mediated Ewing sarcoma oncogenesis." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1523384027382108.
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Kabbout, Mohamed Nazih. "ETS1 AND ETS2 ROLE IN RAS ONCOGENIC TRANSFORMATION IN MOUSE EMBRYONIC FIBROBLASTS." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1275408102.
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Sandberg, Eric M. "Jak2 tyrosine kinase new insights regarding structure, function, and pharmacology /." [Gainesville, Fla.] : University of Florida, 2004. http://purl.fcla.edu/fcla/etd/UFE0006882.
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Thesis (Ph.D.)--University of Florida, 2004.Typescript. Title from title page of source document. Document formatted into pages; contains 118 pages. Includes Vita. Includes bibliographical references.
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Talarico, Alexander Phillip. "Myf5 Does Not Induce Apoptosis In Skeletal Myoblasts But Is Regulated By Oncogenic Ras Expression." Cleveland State University / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=csu1234402667.
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Taffany, David Austin. "Ets2 and PU.1 Cooperatively Regulate Key Oncogenic Pathways in Tumor-Associated Macrophages." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1409014508.
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Rambani, Komal. "A genome wide screen in C. elegans identifies cell non-autonomous regulators of oncogenic Ras mediated over-proliferation." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1461288131.
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Davis, Keira C. "Characterization of Zic2 as an Oncoprotein in Prostate Cancer." DigitalCommons@Robert W. Woodruff Library, Atlanta University Center, 2017. http://digitalcommons.auctr.edu/cauetds/71.
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The field of prostate cancer research is in need of biological markers that predict which cancers do not need treatment, those that can be treated successfully with a localized treatment and more specific cases in which patients are likely to have an aggressive form of cancer that will require more aggressive surgical and chemotherapeutic treatments. ZIC2 is one of five members of a family of proteins that play critical roles in neural crest and mesoderm growth in normal embryonic brain development and in the adult cerebellum of vertebrates. Found throughout the animal kingdom, ZIC1-5 genes encode five distinct ZIC proteins containing five highly conserved C2H2-type zinc finger motifs whose structural integrity is important in carrying out its function as a transcription factor. We hypothesize that ZIC2 has functional significance at the molecular and cellular levels in the initiation of prostate adenocarcinoma (PRAD) and the progression to metastatic and/or castration resistant prostate cancer (CRPC). Bioinformatic predictions suggest that the function of ZIC2 is regulated by post-translational modifications, such as phosphorylation, ubiquitination and sumoylation. This proposal further outlines the research hypothesis for investigating the role of ZIC2 in prostate cancer progression and the effects of the post-translational modification, ubiquitination, on the loss or gain of function of ZIC2.
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Farrington, Caroline Cain. "TARGETED DEGRADATION OF THE MYC ONCOGENE USING PP2AB56ALPHASELECTIVE SMALL MOLECULE MODULATORS OF PROTEINPHOSPHATASE 2A AS A THERAPEUTIC STRATEGY FOR TREATING MYCDRIVENCANCERS." Case Western Reserve University School of Graduate Studies / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1579905487094187.
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Majumdar, Avijit. "Regulation of the activation and activity of the extra-cellular signal regulated kinases 1 & 2 MAP kinase pathway by eukaryotic initiation factor 2 associated glycoprotein p67." [Kent, Ohio] : Kent State University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=kent1209000031.
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Thesis (Ph.D.)--Kent State University, 2008.Title from PDF t.p. (viewed Jan. 26, 2010). Advisor: Bansidhar Datta. Keywords: p67, ERK1, ERK2, oncogenic KRasV12, tumor suppressor. Includes bibliographical references (p. 143-160).
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Morris, Benjamin L. "Understanding and targeting the C-terminal Binding Protein (CtBP) substrate-binding domain for cancer therapeutic development." VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4434.
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Cancer involves the dysregulated proliferation and growth of cells throughout the body. C-terminal binding proteins (CtBP) 1 and 2 are transcriptional co-regulators upregulated in several cancers, including breast, colorectal, and ovarian tumors. CtBPs drive oncogenic properties, including migration, invasion, proliferation, and survival, in part through repression of tumor suppressor genes. CtBPs encode an intrinsic dehydrogenase activity, utilizing intracellular NADH concentrations and the substrate 4-methylthio-2-oxobutyric acid (MTOB), to regulate the recruitment of transcriptional regulatory complexes. High levels of MTOB inhibit CtBP dehydrogenase function and induce cytotoxicity among cancer cells in a CtBP-dependent manner. While encouraging, a good therapeutic would utilize >100-fold lower concentrations. Therefore, we endeavored to design better CtBP-specific therapeutics. The best of these drugs, 3-Cl and 4-Cl HIPP, exhibit nanomolar enzymatic inhibition and micromolar cytotoxicity and showed that CtBP enzymatic function is subject to allosteric interactions. Additionally, the function of the substrate-binding domain has yet to be examined in context of CtBP’s oncogenic activity. To this end, we created several point mutations in the CtBP substrate-binding pocket and determined key residues for CtBP’s enzymatic activity. We found that a conserved tryptophan in the catalytic domain is imperative for function and unique to CtBPs among dehydrogenases. Knowledge of this and other residues allows the directed synthesis of drugs with increased potency and higher CtBP specificity. Early work interrogated the importance of these residues in cell migration. Taken together, this work addresses the utility of the CtBP substrate-binding domain as a target for cancer therapeutics.
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Schneider, Dorit [Verfasser], Dirk [Akademischer Betreuer] Heckl, Jan-Henning [Akademischer Betreuer] Klusmann, Michael [Akademischer Betreuer] Heuser, and Gudrun [Akademischer Betreuer] Göhring. "Deciphering the oncogenic network of PRC2 loss guided leukemogenesis / Dorit Schneider ; Akademische Betreuer: Dirk Heckl, Jan-Henning Klusmann, Michael Heuser, Gudrun Göhring ; Hannover Biomedical Research School, Klinik für Pädiatrische Hämatologie und Onkologie." Hannover : Bibliothek der Medizinischen Hochschule Hannover, 2020. http://d-nb.info/1218185090/34.
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Rush, Craig M. "Characterization of MAX and FOXA2 mutations unique to endometrial cancer." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1542204873523922.
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Ataman, Bulent. "The Molecular Mechanisms of Activity-Dependent Wingless (Wg)/Wnt Signaling at a Drosophila Glutamatergic Synapse: a Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/353.
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Synaptic plasticity, the ability of synapses to change in strength, underlies complex brain functions such as learning and memory, yet little is known about the precise molecular mechanisms and downstream signaling pathways involved. The major goal of my doctoral thesis was to understand these molecular mechanisms and cellular processes underlying synaptic plasticity using the Drosophilalarval neuromuscular junction (NMJ) as a model system.
My work centered on a signaling pathway, the Wg/Wnt signaling pathway, which was found to be crucial for activity-driven synapse formation. The Wg/Wnt family of secreted proteins, besides its well-characterized roles in embryonic patterning, cell growth and cancer, is beginning to be recognized as a pivotal player during synaptic differentiation and plasticity in the brain. At the DrosophilaNMJ, the Wnt-1 homolog Wingless (Wg) is secreted from presynaptic terminals and binds to Frizzled-2 (DFz2) receptors in the postsynaptic muscle. Perturbations in Wg signaling lead to poorly differentiated NMJs, containing synaptic sites that lack both neurotransmitter release sites and postsynaptic structures. In collaboration with other members of the Budnik lab, I set out to unravel the mechanisms by which Wg regulates synapse differentiation. We identified a novel transduction pathway that provides communication between the postsynaptic membrane and the nucleus, and which is responsible for proper synapse development. In this novel Frizzled Nuclear Import (FNI) pathway, the DFz2 receptor is internalized and transported towards the nucleus. The C-terminus of DFz2 is subsequently cleaved and imported into the postsynaptic nucleus for potential transcriptional regulation of synapse development (Mathews, Ataman, et al. Science (2005) 310:1344).
My studies also centered on the genetic analysis of Glutamate Receptor (GluR) Interacting Protein (dGRIP), which in mammals has been suggested to regulate the localization of GluRs and more recently, synapse development. I generated mutations in the gene, transgenic strains carrying a dGRIP-RNAi and fluorescently tagged dGRIP, and antibodies against the protein. Remarkably, I found dgrip mutants had synaptic phenotypes that closely resembled those in mutations altering the FNI pathway. Through the genetic analysis of dgrip and components of the FNI pathway, immunoprecipitation studies, electron microscopy, in vivotrafficking assays, time-lapse imaging, and yeast two-hybrid assays, I demonstrated that dGRIP had a hitherto unknown role as an essential component of the FNI pathway. dGRIP was found in trafficking vesicles that contain internalized DFz2. Further, DFz2 and dGRIP likely interact directly. Through the use of pulse chase experiments I found that dGRIP is required for the transport of DFz2 from the synapse to the nucleus. These studies thus provided a molecular mechanism by which the Wnt receptor, DFz2, is trafficked from the postsynaptic membrane to the nucleus during synapse development and implicated dGRIP as an essential component of the FNI pathway (Ataman et al. PNAS (2006) 103:7841).
In the final part of my dissertation, I concentrated on understanding the mechanisms by which neuronal activity regulates synapse formation, and the role of the Wnt pathway in this process. I found that acute changes in patterned activity lead to rapid modifications in synaptic structure and function, resulting in the formation of undifferentiated synaptic sites and to the potentiation of spontaneous neurotransmitter release. I also found that these rapid modifications required a bidirectional Wg transduction pathway. Evoked activity induced Wg release from synaptic sites, which stimulated both the postsynaptic FNI pathway, as well as an alternative presynaptic Wg pathway involving GSK-3ß/Shaggy. I suggest that the concurrent activation of these alternative pathways by the same ligand is employed as a mechanism for the simultaneous and coordinated assembly of the pre- and postsynaptic apparatus during activity-dependent synapse remodeling (Ataman et al. Neuron (2008) in press).
In summary, my thesis work identified and characterized a previously unrecognized synaptic Wg/Wnt transduction pathway. Further, it established a mechanistic link between activity-dependent synaptic plasticity and bidirectional Wg/Wnt signaling. These findings provide novel mechanistic insight into synaptic plasticity.
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O'Neil, Jennifer Elinor. "Mechanisms of TAL1 Induced Leukemia in Mice: A Dissertation." eScholarship@UMMS, 2004. https://escholarship.umassmed.edu/gsbs_diss/70.
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Activation of the basic helix-loop-helix (bHLH) gene TAL1 is the most common genetic event seen in both childhood and adult T cell acute lymphoblastic leukemia (T-ALL). Despite recent success in treating T-ALL patients, TAL1 patients do not respond well to current therapies. In hopes of leading the way to better therapies for these patients, we have sought to determine the mechanism(s) of Tal1 induced leukemia in mice. By generating a DNA-binding mutant Tal1 transgenic mouse we have determined that the DNA binding activity of Tal1 is not required to induce leukemia. We have also shown that Tal1 expression in the thymus affects thymocyte development and survival. We demonstrate that Tal1 heterodimerizes with the class I bHLH proteins E47 and HEB in our mouse models of TAL1 induced leukemia. Severe thymocyte differentiation arrest and disease acceleration in Tal1/E2A+/- and Tal1/HEB+/- mice provides genetic evidence that Tal1 causes leukemia by inhibiting the function of the transcriptional activators E47 and HEB which have been previously shown to be important in T cell development. In pre-leukemic Tal1 thymocytes, we find the co-repressor mSin3A/HDAC1 bound to the CD4 enhancer, whereas an E47/HEB/p300 complex is detected in wild type thymocytes. Furthermore, mouse Tal1 tumors are sensitive to pharmacologic inhibition of HDAC and undergo apoptosis. These data demonstrate that Tal1 induces T cell leukemia by repressing the transcriptional activity of E47/HEB and suggests that HDAC inhibitors may prove efficacious in T-ALL patients that express TAL1.
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Padmanabhan, Srivatsan. "Worming to Complete the Insulin/IGF-1 Signaling Cascade: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/420.
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The insulin/IGF-1 signaling (IIS) was initially identified in C. elegansto control a developmental phenotype called dauer. Subsequently, it was realized that lifespan was extended by mutations in this pathway and became an intense focus of study. The IIS pathway regulates growth, metabolism and longevity across phylogeny and plays important roles in human disease such as cancer and diabetes. Given the large number of cellular processes that this pathway controls, understanding the regulatory mechanisms that modulate insulin/IGF-1 signaling is of paramount importance.
IIS signaling is a very well-studied kinase cascade but few phosphatases in the pathway are known. Identification of these phosphatases, especially those that counteract the activity of the kinases, would provide a better insight into the regulation of this critical pathway. Study of serine/threonine phosphatases is hampered by the lack of appropriate reagents.
In Chapter II, we discuss the design and results of an RNAi screen of serine/threonine phosphatases performed in C. elegans using dauer formation as a phenotypic output. We identified several strong regulators of dauer formation and in Chapter III, proceed to characterize one of the top candidates of our screen, pptr-1. We show that pptr-1 regulates the IIS and thereby affects lifespan, development and metabolism in C .elegans.
pptr-1gene encodes a protein with high homology to the mammalian B56 family of PP2A regulatory subunits. PP2A is a ubiquitously expressed phosphatase that is involved in multiple cellular processes whose specificity determined by its association with distinct regulatory subunits.
Our studies using C. elegans provides mechanistic insight into how the PP2A regulatory subunit PPTR-1 specifically modulates AKT-1 activity by regulating its phosphorylation status in the context of a whole organism. Furthermore, we show that this mechanism of regulation is conserved in mammals.
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Smith, Jordan L. "Reversing Cancer Cell Fate: Driving Therapeutic Differentiation of Hepatoblastoma to Functional Hepatocyte-Like Cells." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1067.
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Background & Aims: Despite advances in surgical care and chemotherapeutic regimens, the five-year survival rate for Stage IV Hepatoblastoma (HB), the predominant pediatric liver tumor, remains at 27%. YAP1 and β-Catenin co-activation occurs in 80% of children’s HB; however, a lack of conditional genetic models precludes exploration of tumor maintenance and therapeutic targets. Thus, the clinical need for a targeted therapy remains unmet. Given the predominance of YAP1 and β-catenin activation in children’s tumors, I sought to evaluate YAP1 as a therapeutic target in HB.
Approach & Results: Herein, I engineered the first conditional murine model of HB using hydrodynamic injection to deliver transposon plasmids encoding inducible YAP1S127A, constitutive β-CateninDelN90, and a luciferase reporter to murine liver. Tumor regression was evaluated using in vivo bioluminescent imaging, and tumor landscape characterized using RNA sequencing, ATAC sequencing and DNA foot-printing. Here I show that YAP1 withdrawal in mice mediates >90% tumor regression with survival for 230+ days. Mechanistically, YAP1 withdrawal promotes apoptosis in a subset of tumor cells and in remaining cells induces a cell fate switch driving therapeutic differentiation of HB tumors into Ki-67 negative “hbHep cells.” hbHep cells have hepatocyte-like morphology and partially restored mature hepatocyte gene expression. YAP1 withdrawal drives formation of hbHeps by modulating liver differentiation transcription factor (TF) occupancy. Indeed, tumor-derived hbHeps, consistent with their reprogrammed transcriptional landscape, regain partial hepatocyte function and can rescue liver damage in mice.
Conclusions: YAP1 withdrawal, without modulation of oncogenic β-Catenin, significantly regresses hepatoblastoma, providing the first in vivo data to support YAP1 as a therapeutic target for HB. Modulating YAP1 expression alone is sufficient to drive long-term regression in hepatoblastoma because it promotes cell death in a subset of tumor cells and modulates transcription factor occupancy to reverse the fate of residual tumor cells to mimic functional hepatocytes.
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Gannon, Hugh S. "Mdm2-p53 Signaling in Tissue Homeostasis and the DNA Damage Response: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/631.
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The p53 transcription factor responds to various cellular stressors by regulating the expression of numerous target genes involved in cellular processes such as cell cycle arrest, apoptosis, and senescence. As these downstream pathways are harmful to the growth and development of normal cells when prolonged or deregulated, p53 activity needs to be under tight regulatory control. The Mdm2 oncoprotein is the chief negative regulator of p53, and many mouse models have demonstrated that absence of Mdm2 expression leads to constitutive p53 activation in a variety of cell types. While unregulated p53 can be deleterious to cells, functional p53 is essential for tumor suppression, as many human cancers harbor p53 mutations and p53 knockout mice rapidly develop spontaneous tumors. Therefore, the mechanisms that control p53 regulation by Mdm2 are critical to ensure p53 activity in the appropriate cellular context.
Many genetically engineered mouse models have been created to analyze p53 and Mdm2 functions and these studies have yielded valuable insights into their physiological roles. This dissertation will describe the generation and characterization of novel mutant Mdm2 mouse models and their use to interrogate the roles of p53-Mdm2 signaling in tissue homeostasis and cell stress responses. Deletion of Mdm2 in epidermal progenitor cells of the skin and hair follicles resulted in progressive hair loss and decreased skin integrity, phenotypes that are characteristic of premature aging. Furthermore, p53 protein levels, p53 target gene expression, and cellular senescence were all upregulated in the skins of these mice, and epidermal stem cell numbers and function were diminished. These results indicate that Mdm2 is necessary to limit p53 activity in adult tissues to ensure normal stem cell function.
Additional mouse models used to determine the role of Mdm2 phosphorylation will also be presented. DNA damage triggers an extensive cellular response, including activation of the ATM kinase. ATM activity is necessary for p53 protein stabilization and, therefore, p53 activation, but in vivo evidence suggests that phosphorylation of p53 itself had little effect on p53 stability. ATM was previously shown to phosphorylate MDM2 at serine residue 395 (394 in mice), and we generated knock-in mutant mouse models to study the role of this posttranslational modification in vivo. Absence of this phosphorylation site led to greatly diminished p53 stability and function in response to γ-irradiation and increased spontaneous tumorigenesis in mice. Conversely, a phosphomimic model demonstrated prolonged p53 activation in cells treated with γ-irradiation, which revealed that phosphorylation of this Mdm2 residue controls the duration of the DNA damage response. Therefore, these mouse models have uncovered new roles for the p53-Mdm2 regulatory axis in vivo and will be useful reagents in future studies of posttranslational modifications in oncogene and DNA damage-induced tumorigenesis.
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Litteral, Vaughn. "mdm2 Amplification in NIH3T3L1 Preadipocytes Leads to Mdm2 Elevation in Terminal Adipogenesis." Wright State University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=wright1216825497.
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Bei, Yanxia. "Analysis of Polarity Signaling in Both Early Embryogenesis and Germline Development in C. Elegans: A Dissertation." eScholarship@UMMS, 2005. https://escholarship.umassmed.edu/gsbs_diss/147.
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In a 4-cell C. elegans embryo the ventral blastomere EMS requires polarity signaling from its posterior sister cell, P2. This signaling event enables EMS to orient its division spindle along the anterior-posterior (A/P) axis and to specify the endoderm fate of its posterior daughter cell, E.
Wnt pathway components have been implicated in mediating P2/EMS signaling. However, no single mutants or various mutant combinations of the Wnt pathway components disrupt EMS polarity completely. Here we describe the identification of a pathway that is defined by two tyrosine kinase related proteins, SRC-1 and MES-1, which function in parallel with Wnt signaling to specify endoderm and to orient the division axis of EMS. We show that SRC-1, a C. elegans homolog of c-Src, functions downstream of MES-1 to specifically enhance phosphotyrosine accumulation at the P2/EMS junction in order to control cell fate and mitotic spindle orientation in both the P2 and EMS cells.
In the canonical Wnt pathway, GSK-3 is conserved across species and acts as a negative regulator. However, in C. elegans we find that GSK-3 functions in a positive manner and in parallel with other components in the Wnt pathway to specify endoderm during embryogenesis. In addition, we also show that GSK-3 regulates C. elegans germline development, a function of GSK-3 that is not associated with Wnt signaling. It is required for the differentiation of somatic gonadal cells as well as the regulation of meiotic cell cycle in germ cells. Our results indicate that GSK-3 modulates multiple signaling pathways to regulate both embryogenesis and germline development in C. elegans.
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Zhang, Yan. "Probing Cancer Targets and Therapeutic Mechanisms using Small Molecules." Thesis, 2019. https://doi.org/10.7916/D8WM2XC5.
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Small molecules are a powerful tool to illuminate biological mechanisms and assist in the identification and validation of therapeutic targets. KRAS is the single most frequently mutated oncogene in human cancer, with particularly high mutation frequencies observed in pancreas (95%), colon (45%), and lung (35%) cancer. However, despite three decades of effort, there is no clinical viable KRAS cancer therapy. The first part of this thesis focuses on exploring the potential of directly targeting the KRAS nucleotide binding site. Directly targeting oncogenic KRAS with small molecules in the nucleotide-binding site has had limited success due to the high affinity of KRAS for nucleotide GTP and the high cellular concentration of GTP. The strategy of generating engineered KRAS allele based on shape and covalent complementarity was exploited herein to address this challenge. Using fragment-based small molecule design, a cell-membrane-permeable covalent inhibitor able to irreversibly modify the engineered nucleotide-binding site of KRAS was developed. The second part of this thesis describes the investigation of the therapeutic potential of imidazole ketone erastin (IKE), a small molecule inhibitor of the cystine/glutamate antiporter system xc–, in a subcutaneous xenograft model of Diffuse Large B Cell Lymphoma (DLBCL). A biodegradable polyethylene glycol-poly(lactic-co-glycolic acid) nanoparticle formulation was employed to aid in the delivery of IKE to cancer cells in vivo. This IKE nanoparticle system showed improved tumor accumulation and therapeutic index relative to free IKE, indicating its potential for treating DLBCL. The final part of this thesis describes the study of lipid metabolism features of ferroptotic cell death using quantitative reverse transcription PCR (RT-qPCR) and mass spectrometry-based lipidomic analysis. In summary, this work illustrates how chemistry and chemical biology approaches can supplement existing efforts towards the design and discovery of new drugs for challenging targets, as well as aid in the study of therapeutic mechanisms.
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Ranahan, William P. "The oncogenic properties of Amot80 in mammary epithelia." Thesis, 2014. http://hdl.handle.net/1805/4082.
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Indiana University-Purdue University Indianapolis (IUPUI)While breast cancer is the second most commonly diagnosed cancer worldwide, its causes and natural history are not well defined. The female mammary organ is unique in that it does not reach full maturity until the lactation cycle following pregnancy. This cycle entails extensive growth and reorganization of the primitive epithelial ductal network. Following lactation, these same epithelial cells undergo an equally extensive program of apoptosis and involution. The mammary gland's sensitivity to pro-growth and pro-apoptotic signals may partly explain its proclivity to develop cancers. For epithelial cells to become transformed they must lose intracellular organization known as polarity as differentiated epithelial tissues are refractory to aberrant growth. One essential component of epithelial to mesenchymal transition is the intrinsic capacity of cells to repurpose polarity constituents to promote growth. Recently, a novel mechanism of organ size control has been shown to repurpose the apical junctional associated protein Yap into the nucleus where it functions as a transcriptional coactivator promoting growth and dedifferentiation. The focus of my work has been on a family of adaptor proteins termed Amots that have been shown to scaffold Yap and inhibit growth signaling. Specifically, I have shown that the 80KDa form of Amot, termed Amot80, acts as a dominant negative to the other Amot proteins to promote cell growth while reducing cell differentiation. Amot80 was found to promote the prolonged activation of MAPK signaling. Further, Amot80 expression was also found to enhance the transcriptional activity of Yap. This effect likely underlies the ability of Amot80 to drive disorganized overgrowth of MCF10A cells grown in Matrigel̈™. Overall, these data suggest a mechanism whereby the balance of Amot proteins controls the equilibrium between growth and differentiation within mammary epithelial tissues.
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Adler, Jacob J. "The inhibition of mammary epithelial cell growth by the long isoform of Angiomotin." Thesis, 2014. http://hdl.handle.net/1805/4600.
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Indiana University-Purdue University Indianapolis (IUPUI)Mammary ductal epithelial cell growth is controlled by microenvironmental signals in serum under both normal physiological settings and during breast cancer progression. Importantly, the effects of several of these microenvironmental signals are mediated by the activities of the tumor suppressor protein kinases of the Hippo pathway. Canonically, Hippo protein kinases inhibit cellular growth through the phosphorylation and inactivation of the oncogenic transcriptional co-activator Yes-Associated Protein (YAP). This study defines an alternative mechanism whereby Hippo protein kinases induce growth arrest via the phosphorylation of the long isoform of Angiomotin (Amot130). Specifically, serum starvation is found to activate the Hippo protein kinase, Large Tumor Suppressor (LATS), which phosphorylates the adapter protein Amot130 at serine-175. Importantly, wild-type Amot130 potently inhibits mammary epithelial cell growth, unlike the Amot130 serine-175 to alanine mutant, which cannot be phosphorylated at this residue. The growth-arrested phenotype of Amot130 is likely a result of its mechanistic response to LATS signaling. Specifically, LATS activity promotes the association of Amot130 with the ubiquitin ligase Atrophin-1 Interacting Protein 4 (AIP4). As a consequence, the Amot130-AIP4 complex amplifies LATS tumor suppressive signaling by stabilizing LATS protein steady state levels via preventing AIP4-targeted degradation of LATS. Additionally, AIP4 binding to Amot130 leads to the ubiquitination and stabilization of Amot130. In turn, the Amot130-AIP4 complex signals the ubiquitination and degradation of YAP. This inhibition of YAP activity by Amot130 requires both AIP4 and the ability of Amot130 to be phosphorylated by LATS. Together, these findings significantly modify the current view that the phosphorylation of YAP by Hippo protein kinases is sufficient for YAP inhibition and cellular growth arrest. Based upon these results, the inhibition of cellular growth in the absence of serum more accurately involves the stabilization of Amot130 and LATS, which together inhibit YAP activity and mammary epithelial cell growth.
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Bai, Yunpeng. "Understanding the biological function of phosphatases of regenerating liver, from biochemistry to physiology." Thesis, 2014. http://hdl.handle.net/1805/5675.
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Indiana University-Purdue University Indianapolis (IUPUI)Phosphatases of regenerating liver, consisting of PRL-1, PRL-2 and PRL-3, belong to a novel protein tyrosine phosphatases subfamily, whose overexpression promotes cell proliferation, migration and invasion and contributes to tumorigenesis and metastasis. However, although great efforts have been made to uncover the biological function of PRLs, limited knowledge is available on the underlying mechanism of PRLs’ actions, therapeutic value by targeting PRLs, as well as the physiological function of PRLs in vivo. To answer these questions, we first screened a phage display library and identified p115 RhoGAP as a novel PRL-1 binding partner. Mechanistically, we demonstrated that PRL-1 activates RhoA and ERK1/2 by decreasing the association between active RhoA with GAP domain of p115 RhoGAP, and displacing MEKK1 from the SH3 domain of p115 RhoGAP, respectively, leading to enhanced cell proliferation and migration. Secondly, structure-based virtual screening was employed to discover small molecule inhibitors blocking PRL-1 trimer formation which has been suggested to play an important role for PRL-1 mediated oncogenesis. We identified Cmpd-43 as a novel PRL-1 trimer disruptor. Structural study demonstrated the binding mode of PRL-1 with the trimer disruptor. Most importantly, cellular data revealed that Cmpd-43 inhibited PRL-1 induced cell proliferation and migration in breast cancer cell line MDA-MB-231 and lung cancer cell line H1299. Finally, in order to investigate the physiological function of PRLs, we generated mouse knockout models for Prl-1, Prl-2 and Prl-3. Although mice deficient for Prl-1 and Prl-3 were normally developed, Prl-2-null mice displayed growth retardation, impaired male reproductive ability and insufficient hematopoiesis. To further investigate the in vivo function of Prl-1, we generated Prl-1-/-/Prl-2+/- and Prl-1+/-/Prl-2-/- mice. Similar to Prl-2 deficient male mice, Prl-1-/-/Prl-2+/- males also have impaired spermatogenesis and reproductivity. More strikingly, Prl-1+/-/Prl-2-/- mice are completely infertile, suggesting that, in addition to PRL-2, PRL-1 also plays an important role in maintaining normal testis function. In summary, these studies demonstrated for the first time that PRL-1 activates ERK1/2 and RhoA through the novel interaction with p115 RhoGAP, targeting PRL-1 trimer interface is a novel anti-cancer therapeutic treatment and both PRL-1 and PRL-2 contribute to spermatogenesis and male mice reproductivity.
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Walls, Chad Daniel. "Functional Insights Into Oncogenic Protein Tyrosine Phosphatases By Mass Spectrometry." Thesis, 2014. http://hdl.handle.net/1805/3879.
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Indiana University-Purdue University Indianapolis (IUPUI)Phosphatase of Regenerating Liver 3 (PRL3) is suspected to be a causative factor toward cellular metastasis when overexpressed. To date, the molecular basis for PRL3 function remains an enigma, justifying the use of 'shot-gun'-style phosphoproteomic strategies to define the PRL3-mediated signaling network. On the basis of aberrant Src tyrosine kinase activation following ectopic PRL3 expression, phosphoproteomic data reveal a signal transduction network downstream of a mitogenic and chemotactic PDGF (α and β), Eph (A2, B3, B4), and Integrin (β1 and β5) receptor array known to be utilized by migratory mesenchymal cells during development and acute wound healing in the adult animal. Tyrosine phosphorylation is present on a multitude of signaling effectors responsible for Rho-family GTPase, PI3K-Akt, Jak-STAT3, and Ras-ERK1/2 pathway activation, linking observations made by the field as a whole under Src as a primary signal transducer. Our phosphoproteomic data paint the most comprehensive picture to date of how PRL3 drives pro-metastatic molecular events through Src activation. The Src-homology 2 (SH2) domain-containing tyrosine phosphatase 2 (SHP2), encoded by the Ptpn11 gene, is a bona-fide proto-oncogene responsible for the activation of the Ras/ERK1/2 pathway following mitogen stimulation. The molecular basis for SHP2 function is pTyr-ligand-mediated alleviation of intramolecular autoinhibition by the N-terminal SH2 domain (N-SH2 domain) upon the PTP catalytic domain. Pathogenic mutations that reside within the interface region between the N-SH2 and PTP domains are postulated to weaken the autoinhibitory interaction leading to SHP2 catalytic activation in the open conformation. Conversely, a subset of mutations resides within the catalytic active site and cause catalytic impairment. These catalytically impaired SHP2 mutants potentiate the pathogenesis of LEOPARD-syndrome (LS), a neuro-cardio-facial-cutaneous (NCFC) syndrome with very similar clinical presentation to related Noonan syndrome (NS), which is known to be caused by gain-of-function (GOF) SHP2 mutants. Here we apply hydrogen-deuterium exchange mass spectrometry (H/DX-MS) to provide direct evidence that LS-associated SHP2 mutations which cause catalytic impairment also weaken the autoinhibitory interaction that the N-SH2 domain makes with the PTP domain. Our H/DX-MS study shows that LS-SHP2 mutants possess a biophysical property that is absolutely required for GOF-effects to be realized, in-vivo.
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Martin, Holly René. "Mechanism of Transformation and Therapeutic Targets for Hematological Neoplasms Harboring Oncogenic KIT Mutation." Thesis, 2014. http://hdl.handle.net/1805/5503.
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Indiana University-Purdue University Indianapolis (IUPUI)Gain-of-function mutations in the KIT receptor tyrosine kinase have been associated with highly malignant human neoplasms. In particular, an acquired somatic mutation at codon 816 in the second catalytic domain of KIT involving an aspartic acid to valine substitution is found in patients with systemic mastocytosis (SM) and acute myeloid leukemia (AML). The presence of this mutation in SM and AML is associated with poor prognosis and overall survival. This mutation changes the conformation of the KIT receptor resulting in altered substrate recognition and constitutive tyrosine autophosphorylation leading to constitutive ligand independent growth. As there are currently no efficacious therapeutic agents against this mutation, this study sought to define novel therapeutic targets that contribute to aberrant signaling downstream from KITD816V that promote transformation of primary hematopoietic stem/progenitor cells in diseases such as AML and SM. This study shows that oncogenic KITD814V (murine homolog) induced myeloproliferative neoplasms (MPN) occurs in the absence of ligand stimulation, and that intracellular tyrosines are important for KITD814V-induced MPN. Among the seven intracellular tyrosines examined, tyrosine 719 alone has a unique role in regulating KITD814V-induced proliferation and survival. Residue tyrosine 719 is vital for activation of the regulatory subunit of phosphatidylinositol 3-kinase (PI3K), p85α, downstream from KITD814V. Downstream effectors of the PI3K signaling pathway, in of leukemic cells bearing KITD814V with an allosteric inhibitor of Pak or its genetic inactivation results in growth repression due to enhanced apoptosis. To assess the role of Rac GEFs in KITD814V induced transformation, EHop-016, an inhibitor of Rac, was used to specifically target Vav1, and found to be a potent inhibitor of human and murine leukemic cell growth. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of MPN and rescued the associated pathology in mice. These studies provide insight on mechanisms and potential novel therapeutic targets for hematological malignancies harboring an oncogenic KIT mutation.
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(5930141), Minervo Perez. "HIGH-THROUGHPUT IDENTIFICATION OF ONCOGENIC TYROSINE KINASE SUBSTRATE PREFERENCES TO IMPROVE METHODS OF DETECTION." Thesis, 2021.
Find full textAbstract:
The use of computational approaches to understand kinase substrate preference has been a powerful tool in the search to develop artificial peptide probes to monitor kinase activity, however, most of these efforts focus on a small portion of the human kinome. The use of high throughput techniques to identify known kinase substrates plays an important role in development of sensitive protein kinase activity assays.
The KINATEST-ID pipeline is an example of a computational tool that uses known kinase substrate sequence information to identify kinase substrate preference. This approach was used to design three artificial substrates for ABL, JAK2 and SRC family kinases. These biosensors were used to design ELISA and lanthanide-based assays to monitor in vitro kinase activity. The KINATEST-ID pipeline relies on a high number of reported kinase substrates to predict artificial substrate sequences, however, not all kinases have the sufficient number of known substrates to make an accurate prediction.
The adaptation of kinase assay linked with phosphoproteomics technique was used to increase the number of known FLT3 kinase variant substrate sequences. Subsequently, a set of data formatting tools were developed to curate the mass spectrometry data to become compatible with a command line version of the KINATEST-ID pipeline modules. This approach was used to design seven pan-FLT3 artificial substrate (FAStides) sequences. The pair of FAStides that were deemed the most sensitive toward FLT3 kinase phosphorylation were assayed in increasing concentrations of clinically relevant tyrosine kinase inhibitors.
To improve the automation of the mass spectrometry data analysis and formatting for use with the KINATEST-ID pipeline, a streamlined process was developed within a bioinformatic platform, GalaxyP. The data formatting tools used to process the FLT3 mass spectrometry data were converted into compatible versions to execute within the GalaxyP framework. This process was used to design four BTK artificial substrates (BAStide) to monitor kinase activity. Additionally, one of the BAStide sequences was designed in the lanthanide chelating motif to develop an antibody-free activity assay for BTK.
Lastly, a multicolored time resolved lanthanide assay was designed by labeling SYK artificial substrate and a SRC family artificial substrate to measure the activity of both kinases in the same kinase reaction. This highlighted the functionality of lanthanide-based time resolved assays for potential multiplexing assay development.