Irx5 is a homeodomain transcription factor that negatively regulates cardiac fast transient outward K+ currents (Ito,f) via the KV4.2 gene and is thereby a major determinant of the transmural repolarization gradient. While Ito,f is invariably reduced in heart disease and changes in Ito,f can modulate both cardiac contractility and hypertrophy, less is known about a functional role of Irx5, and its relationship with Ito,f, in the normal and diseased heart. Here I show that Irx5 plays crucial roles in the regulation of cardiac contractility and proper adaptive hypertrophy. Specifically, Irx5-deficient (Irx5-/-) hearts had reduced cardiac contractility and lacked the normal regional difference in excitation-contraction with decreased action potential duration, Ca2+ transients and myocyte shortening in sub-endocardial, but not sub-epicardial, myocytes. In addition, Irx5-/- mice showed less cardiac hypertrophy, but increased interstitial fibrosis and greater contractility impairment following pressure overload. A defect in hypertrophic responses in Irx5-/- myocardium was confirmed in cultured neonatal mouse ventricular myocytes, exposed to norepinephrine while being restored with Irx5 replacement. Interestingly, studies using mice virtually lacking Ito,f (i.e. KV4.2-deficient) showed that reduced contractility in Irx5-/- mice was completely restored by loss of KV4.2, whereas hypertrophic responses to pressure-overload in hearts remained impaired when both Irx5 and Ito,f were absent. These findings suggest that Irx5 regulates cardiac contractility in an Ito,f-dependent manner while affecting hypertrophy independent of Ito,f. On the other hand, Irx5-ablation attenuated calcineurin (Cn)-induced hypertrophy in hearts and cultured cardiomyocytes, suggesting that the effect of Irx5 on hypertrophy involves the Cn-NFAT signalling cascade. Biochemical assessments further revealed that Irx5 can positively mediate Cn-NFAT activities as well as Nfatc3 and Gata4 expression, and interacts with Nfatc3 and Gata4, suggesting the formation of a transcription complex for hypertrophic gene regulation. Taken together, these studies have identified Irx5 as a vital cardiac transcription factor, important for contractile function of the heart by regulating Ito,f, and compensatory hypertrophic response to biomechanical stress in the heart by affecting the Cn-NFAT (and Gata4) signaling pathway.

Platinum-based chemotherapies are effective agents in the treatment of a wide variety of human cancers. However, patients with recurrent disease can become resistant to platinum-based chemotherapy, leading to low overall survival rates. Activating transcription factor 3 (ATF3) is a stress-inducible gene that is a regulator of cisplatin-induced cytotoxicity. ATF3 protein expression was upregulated after cytotoxic doses of cisplatin treatment in a panel of cell lines. A chromatin immunoprecipitation assay showed that upon treatment with cisplatin, ATF3 directly bound to the CHOP gene promoter and this correlated with an increase in CHOP protein expression. In a 1200 compound library screen performed on cancer cell lines, disulfiram, a dithiocarbamate drug, was identified as an enhancer of the cytotoxic effects of cisplatin. This increased cytotoxic action was likely due to disulfiram and cisplatin’s ability to induce ATF3 independently through two separate mechanisms, namely the MAPK and integrated stress pathways. Furthermore, ATF3 protein and mRNA levels were variable amongst human ovarian and lung cancer tissues, suggesting the potential for basal expression of ATF3 to be predictive of cisplatin treatment response. Thus, understanding ATF3’s role in cisplatin-induced cytotoxicity will lead to novel therapeutic approaches that could improve this drug’s efficacy.

Abiotic stress is one of the major factors that affect food production worldwide and, therefore understanding stress responsive proteins and engineering plants for abiotic stress tolerance is very important. In the present study, the biological role of pea pathogenesis-related 10.4 (PR-10.4; also known as abscisic acid responsive 17; ABR17) in abiotic stress tolerance has been investigated. Our investigation on ribonuclease (RNase) activity of ABR17 suggested that highly conserved histidine-69 and glutamic acid-148 are important for RNase activity. In order to further investigate the biological role(s) of ABR17, transcriptional profiling of pea ABR17-mediated gene expression changes in ABR17-transgenic Arabidopsis thaliana plants was carried out using microarrays. Our results indicated that pea ABR17 modulates many plant growth/development genes most of which are cytokinin (CK) responsive. These results agree very well with previously reported enhanced endogenous CKs in these transgenic plants. However, no significant changes in transcript abundance of CK biosynthetic genes were observed between transgenic and wild-type plants, suggesting an alternate source of CK in ABR17-transgenic plants. It is speculated that ABR17 may act as either a CK reservoir (through its reported CK binding property) or may be responsible for isopentenylated-tRNA degradation (through its demonstrated RNase activity) thereby increasing endogenous CK pools. Furthermore, microarray analysis of salinity stressed ABR17-Arabidopsis indicated that ABR17 modulates many stress responsive genes that included four putative AP2 family genes (RAP2.6-At1g43160, RAP2.6L-At5g13330, DREB26-At1g21910 and DREB19-At2g38340). Functional characterization of these genes suggested that they are transcription factors and they play very important roles in abiotic stress response in addition to growth and development. Moreover, overexpression of RAP2.6L and DREB19 genes enhanced salinity and drought tolerance in Arabidopsis. Taken together, our results suggest that pea ABR17 proteins are important in abiotic stress responses as they may act as source of enhanced CKs and they may also modulate expression of stress responsive genes to enhance stress tolerance in plants. However, additional research aimed at deciphering the links between ABR17 and CK biosynthesis as well as the mechanism of ABR17-mediated gene expression changes should be conducted in order to get more insights into the biological roles of PR10 proteins in planta. / Plant Science

Forkhead (Fox) proteins are transcription factors that function in many processes including development, metabolism and cell cycle regulation. This gene family is divided into subfamilies that appear to originate from a common ancestor. I have identified the evolutionary selection pressures acting on individual amino acid positions in the FoxA, FoxC, FoxD, FoxI, FoxO and FoxP subfamilies. The patterns of selection observed allowed for the prediction of residue function and identification of residues that differentiate orthologs and paralogs. The subfamily structure and negative selection found within the subfamilies indicates that after gene duplication, differentiation of subfamilies through amino acid changes and subsequent negative selection on these changes has occurred. Meanwhile, the observed neutral changes and positive selection allow for further protein differentiation. Within the FoxC subfamily, positive selection was identified at one amino acid site in the inhibitory domain. Mutation of this site in FOXC1 alters transactivation activity and the effects of mutants on transactivation activity are different on different reporters. The mutant effects were consistent with those of known disease causing mutations, supporting the predicted positive selection. The inhibitory domain is known to function in reducing FOXC1 transactivation activity and influences protein stability. Here I additionally show that loss of the inhibitory domain and mutation of the positively selected site can reduce FOXC1 DNA binding. Co-transfection of FOXC1 and TLE4, a repressor protein that can potentially bind to the inhibitory domain, was shown to increase FOXC1 transactivation activity. The effects of a novel disease causing FOXC1 inhibitory domain mutation on FOXC1 function were also assessed. The mutation reduced FOXC1 transactivation activity and increased protein half-life both of which may lead to disease. Regulation of FOXC1 activity is critical for normal function and this work has furthered our knowledge of how the inhibitory domain influences FOXC1 activity. I have provided biological evidence for the theory that positive selection acts at the amino acid level to optimize protein function. I have also shown that both changes in transcription factor proteins and the cis-regulatory region of target genes have the potential to contribute to evolutionary adaptation.

B cell development is a carefully orchestrated process involving many transcription factors acting in concert with cytokine signals, particularly IL-7. The transcription factor c-Myb has long been implicated in B cell development, however surprisingly little is known about the function of c-Myb in B cell progenitors. I have used several mouse models of c-Myb deficiency to investigate the role of c-Myb in the B cell lineage. Conditional deletion of c-Myb in early B cell progenitors using mb-1Cre (c MybΔmb1/Δmb1) leads to a striking lack of B cells from the pre-pro-B cell stage onwards, demonstrating that c-Myb is absolutely required for B cell development. Mice homozygous for a hypomorphic allele of c-Myb (c MybPlt4/Plt4) also display a severe reduction in B cells; in these mice, defects in lymphoid development can be detected within the multipotent progenitor compartment of bone marrow. c-Myb activates transcription via coactivator proteins, particularly CBP and p300. Mice bearing a point mutation in p300 (p300Plt6/Plt6) that inhibits the interaction of p300 with c Myb display a partial block in B cell development, highlighting the importance of the c Myb-p300 complex for B cell development. Together, these mice demonstrate that c-Myb regulates B cell development by functioning both in multipotent progenitor cells and directly in B cell progenitors. In addition, I show that the B-lymphopenia in c-Myb deficient mice is related to a profound defect in IL-7 signalling. IL-7 normally stimulates the proliferation, survival and differentiation of B cell progenitors, however pro-B cells from c-MybPlt4/Plt4 and c MybΔmb1/Δmb1 mice fail to respond to IL 7. Expression of the IL-7Rα chain is reduced on pro-B cells from c MybPlt4/Plt4 and c-MybΔmb1/Δmb1 mice, suggesting that Il7r may be a c-Myb target gene in B cells. Reporter gene assays show that c-Myb can activate the Il7r promoter in synergy with the transcription factor Pu.1. Overall, this work demonstrates that c-Myb is essential for early B cell development and plays a critical role in linking cytokine signals to the transcription factor networks in B cell progenitors.

Thesis (Ph.D.). / Written for the Dept. of Medicine. Title from title page of PDF (viewed 2008/05/09). Includes bibliographical references.

Thesis (Ph.D.)--University of South Florida, 2001. / Includes vita. Includes bibliographical references (leaves 80-104). Also available online.

Thesis (Ph.D. in Cancer Biology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 182-210). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Molecular Biology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 185-214). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 152 pages. Includes Vita. Includes bibliographical references.