Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 114-125). Also available on the World Wide Web.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 109-119).

Understanding the specific mechanisms regulating chemotherapeutic drug anti-cancer activities will uncover novel strategies to enhance the efficacy of these drugs in clinical settings. Activating Transcription Factor 3 (ATF3) is a stress inducible gene whose expression has been associated with survival outcomes in cancer models. This study characterizes the chemotherapeutic drugs, cisplatin and Histone Deacetylase Inhibitor (HDACi), M344 as novel inducers of ATF3 expression. Cisplatin is a DNA damaging agent widely used in various tumour types including lung, head and neck, and ovarian carcinomas. The HDAC inhibitor, SAHA, has recently been approved as a single agent in the treatment of subcutaneous T-cell lymphoma and HDACis themselves show potential for synergistic anti-cancer effects when used in combination with established chemotherapeutic drugs, including cisplatin. This study evaluates the mechanisms by which cisplatin and HDACi induce ATF3, as well as the role ATF3 plays as a mediator of cisplatin-induced cytotoxicity and the enhanced cytotoxicity between HDACi and cisplatin in combination. In this study, we demonstrate that cytotoxic doses of cisplatin and carboplatin consistently induced ATF3 expression in a panel of human tumour derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response (ISR) independent mechanism, all previously implicated in stress mediated ATF3 induction. Analysis of MAPKinase pathway involvement in ATF3 induction by cisplatin revealed a MAPKinase dependent mechanism. Cisplatin treatment, in combination with specific inhibitors to each MAPKinase pathway (JNK, ERK and p38) resulted in decreased ATF3 induction at the protein level. MAPKinase pathway inhibition led to decreased ATF3 mRNA expression and a reduction in the cytotoxic effects of cisplatin as measured by MTT cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific shRNAs also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3 -/- MEFs were shown to be less sensitive to cisplatin induced cytotoxicity as compared with ATF3+/+ MEFs. Taken together, we identified cisplatin as a MAPKinase pathway dependent inducer of ATF3 whose expression regulates in part cisplatin’s cytotoxic effects. Furthermore, we demonstrated that the HDAC inhibitor M344 was also an inducer of ATF3 expression at the protein and mRNA level in the same human derived cancer cell lines. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatment was also shown to enhance the cytotoxic effects of cisplatin in these cancer cell lines. Unlike cisplatin, the mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways. Utilizing ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) M334 induction of ATF3 was shown to depend on the presence of ATF4, a known regulator of ATF3 expression as part of the ISR pathway. HDACi treatment did not affect the level of histone acetylation associated with the ATF3 promoter as determined through Chromatin immunoprecipitation (ChIP) analysis, suggesting that ATF3 induction was not a direct effect of HDACi mediated histone acetylation. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.

Cardiovascular disease continues to be the leading cause of morbidity and mortality worldwide with an estimated 17 million annual deaths. A majority of cases are attributed to disease affecting the vascular system including arterial, venous and lymphatic vessels. Despite progress in understanding the molecular bases of vascular development and disease, the role of chromatin modifying enzymes in vascular processes remains ill defined. Here we show that the histone-modifying enzyme Hdac3 is a critical regulator of lymphatic vascular development. Endothelial specific loss of Hdac3 in mice affects the development of lymphovenous and lymphatic valves resulting in aberrant blood lymph separation, lymphedema and complete lethality. We demonstrate that Hdac3 functions in a flow responsive manner to regulate the expression of Gata2, a transcription factor essential for lymphatic valve development. In response to flow, transcription factors Tal1, Ets1/2 and Gata2 recruit Hdac3 to an evolutionarily conserved intragenic enhancer of Gata2 gene. In turn, Hdac3 recruits p300, a histone acetyl transferase, to render activation of the Gata2 enhancer, and thus promotes Gata2 transcription. Together, our findings demonstrate the molecular basis by which cell extrinsic and intrinsic cues cooperate to regulate lymphatic development.

Abiotic stress leads to a change in the water content of plants. Salinity and osmotic stress affect both the morphology and physiology of plants. Plants have therefore responded to these environmental changes by adapting and tolerating them. The SABP2-interacting proteins (SIP) 428-silenced RNAi transgenic tobacco lines were subjected to various abiotic stresses (salinity, osmotic, and drought). The effect of SIP428-silencing on the tobacco plants subjected to these abiotic stresses was monitored. The results from the root growth data show that the sip428-silenced lines exhibit enhanced tolerance to the stressors compared to the wild-type plants. Interestingly, results of the relative chlorophyll content show no significant difference between the wild-type plants and sip428-silenced transgenic plants. In summary, based on the results presented in this study it could be concluded that SIP428 is a negative regulator of salinity, osmotic and drought stresses. Further studies are required to understand the mechanism.

Abiotic stresses are constantly rising and pose a very high risk to global agricultural productivity and food security. Some plants have evolved several innate pathways for defense against these stresses. Hence, understanding stress signaling pathways can help develop crop plants with higher stress tolerance. The salicylic acid-mediated signaling pathway is important in plants experiencing biotic and abiotic stresses. In previous studies, SABP2-Interacting Protein (SIP-428) has been shown to be a negative regular of plant growth under abiotic stress. This study aimed to investigate the roles of SIP-428 in the ROS signaling of tobacco plants. We investigated transgenic RNAi-silenced lines of SIP-428 and wild-type tobacco plants for the activities of guaiacol peroxidase and catalase enzymes in Mannitol and NaCl-stressed plants for 7 and 14 days. Our results showed that SIP-428 plays a significant role in ROS signaling in Mannitol and NaCl-stressed plants via the activities of guaiacol peroxidase.

No description available.

Global climate change is identified as a major threat to the survival of natural ecosystems. The variations in global climate have gained the attention of researchers worldwide, as these changes negatively affect agriculture by reducing crop productivity and food security.  Projects related to abiotic stress tolerance are significant because they address important challenges facing agriculture and food security, contribute to more sustainable agricultural practices, and advance our understanding of fundamental plant biology. Some plants have defense mechanisms that are activated upon receiving stress stimuli to increase systemic tolerance to abiotic stresses such as heat, light, and cold. Salicylic acid-binding protein 2 (SABP2) from tobacco exhibits a high affinity for salicylic acid (SA) and is an important component in the SA-signaling pathway. SABP2 interacts with other cellular proteins to initiate downstream signaling and activate responses leading to resistance. Several SABP2-interacting proteins (SIP), including SIP-428, have been identified. The main goal of this proposed research is to determine the role of SIP-428 in mediating environmental stresses. SIP-428 is a SIR2-type non-histone deacetylase enzyme. De/acetylation is a common post-translational modification of proteins in eukaryotes. Since SIP-428 is a SABP2-interacting protein, it is involved in plant immune signaling. To determine the role of SIP-428 in plant physiology, it was biochemically characterized, and transgenic tobacco plants silenced in SIP-428 expression were previously generated and analyzed. Transgenic tobacco plants overexpressing SIP-428 were also generated. These lines expressed SIP428 at higher levels upon treatment with estradiol. Transgenic tobacco that overexpresses SIP-428 has been used in this study. To test the role of SIP428 in abiotic stress, the transgenic plants will be treated with abiotic stress-inducing chemicals, e.g. NaCl (salinity stress), mannitol (osmotic stress), and PEG6000 (drought stress). The treated seedlings will be allowed to grow for a specific time (1-2 weeks). The expression of SIP-428 will be monitored by western blotting (using anti-myc antibodies). The effects of SIP-428 expression on abiotic stress tolerance will be investigated biochemically by examining the activities of antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX). Additionally, gene expression analysis will also be conducted to determine the expression of antioxidant genes.