This thesis explores how basic region/leucine zipper (bZIP) transcription factors target gene regulatory sequences. The GCN4 bZIP binds to more than one target site [CRE (TGACGTCA) and cognate AP-1 (TGACTCA)] and exhibits flexibility in -helical structure. These observations suggest that the GCN4 bZIP can establish sequence-selective DNA binding at noncognate target sites. Studies on such noncognate but sequence-selective binding can provide insights into how bZIP proteins search for and localize to their cognate target sites. This thesis investigates DNA binding by the GCN4 bZIP and its structural and functional mimic, the wild-type (wt) bZIP, at noncognate gene regulatory sequences C/EBP (TTGCGCAA), E-box (CACGTG), HRE (GCACGTAG), XRE1 (TTGCGTGA), and related DNA sequences. These DNA-binding activities are sequence-selective, as confirmed by DNase I footprinting and electrophoretic mobility shift assay (EMSA). Full- and half-site DNA-binding affinities, determined by EMSA titrations, decrease from cognate to noncognate binding. At noncognate target sites, the bZIP proteins form a dimer of -helices, as indicated by circular dichroism (CD) spectroscopy and EMSA. These results demonstrate that the bZIP proteins can establish noncognate but sequence-selective DNA binding, and suggest such DNA binding potentially contributes to structure preorganization and rapid translocation of the bZIP proteins when they search for their cognate target sites, to which they then bind with high affinity. This thesis also indicates a highly dynamic DNA-binding model for the bZIP proteins to establish strong and sequence-selective DNA binding. The C/EBP site includes two 5H-LR (TTGCG) half-sites, each of which comprises two 4-bp subsites. The in vitro and in silico results together demonstrate that the basic region at 5H-LR recognizes the 4-bp subsites alternately as distinct units, which requires it to translocate between the subsites, potentially by sliding or hopping. Taken as a whole, this thesis provides further insights into how bZIP transcription factors accomplish sequence-selective DNA binding.

This thesis explores how basic region/leucine zipper (bZIP) transcription factors target gene regulatory sequences. The GCN4 bZIP binds to more than one target site [CRE (TGACGTCA) and cognate AP-1 (TGACTCA)] and exhibits flexibility in -helical structure. These observations suggest that the GCN4 bZIP can establish sequence-selective DNA binding at noncognate target sites. Studies on such noncognate but sequence-selective binding can provide insights into how bZIP proteins search for and localize to their cognate target sites. This thesis investigates DNA binding by the GCN4 bZIP and its structural and functional mimic, the wild-type (wt) bZIP, at noncognate gene regulatory sequences C/EBP (TTGCGCAA), E-box (CACGTG), HRE (GCACGTAG), XRE1 (TTGCGTGA), and related DNA sequences. These DNA-binding activities are sequence-selective, as confirmed by DNase I footprinting and electrophoretic mobility shift assay (EMSA). Full- and half-site DNA-binding affinities, determined by EMSA titrations, decrease from cognate to noncognate binding. At noncognate target sites, the bZIP proteins form a dimer of -helices, as indicated by circular dichroism (CD) spectroscopy and EMSA. These results demonstrate that the bZIP proteins can establish noncognate but sequence-selective DNA binding, and suggest such DNA binding potentially contributes to structure preorganization and rapid translocation of the bZIP proteins when they search for their cognate target sites, to which they then bind with high affinity. This thesis also indicates a highly dynamic DNA-binding model for the bZIP proteins to establish strong and sequence-selective DNA binding. The C/EBP site includes two 5H-LR (TTGCG) half-sites, each of which comprises two 4-bp subsites. The in vitro and in silico results together demonstrate that the basic region at 5H-LR recognizes the 4-bp subsites alternately as distinct units, which requires it to translocate between the subsites, potentially by sliding or hopping. Taken as a whole, this thesis provides further insights into how bZIP transcription factors accomplish sequence-selective DNA binding.

T-ALL accounts for 15% of childhood leukemias and approximately 60% of patients overexpress TAL1. TAL1/SCL encodes a transcription factor that regulates hematopoiesis by dimerizing with additional transcription factors including E12, E47, and GATA-1. TAL1 has also been found to repress expression of NF-κB1, potentially promoting formation of an NF-κB p65/c-Rel heterodimer that encourages cell survival by up-regulating IAPs and IκB. However, the correlation between TAL1 and p65/c-Rel expression and their effects on downstream targets like IKK, IκB, and other anti-apoptotic proteins is poorly understood. Jurkat cells, expressing TAL1, were treated with TNFα and/or etoposide to induce apoptosis and experiments were performed to assess the expression of proteins of interest. Caspase-8 activity assays were also performed to help delineate the apoptotic signal present in these cells. Determining if interactions between TAL1, NF-κB, and other downstream targets help promote apoptotic resistance will further research into better, more targeted treatments for T-ALL. / Department of Biology

PURPOSE: This phase I dose-escalation study was undertaken to establish the maximum tolerated dose of the sequence-selective minor groove DNA binding agent SJG-136 in patients with advanced solid tumors. The study also investigated antitumor activity and provided pharmacokinetic and pharmacodynamic data. EXPERIMENTAL DESIGN: Sixteen patients were assigned sequentially to escalating doses of SJG-136 (15-240 microg/m(2)) given as a 10-minute i.v. infusion every 21 days. The dose was subsequently reduced in incremental steps to 45 microg/m(2) due to unexpected toxicity. RESULTS: The maximum tolerated dose of SJG-136 was 45 microg/m(2). The main drug-related adverse event was vascular leak syndrome (VLS) characterized by hypoalbuminemia, pleural effusions, ascites, and peripheral edema. Other unexpected adverse events included elevated liver function tests and fatigue. The VLS and liver toxicity had delayed onset and increased in severity with subsequent cycles. Disease stabilization was achieved for >6 weeks in 10 patients; in 2 patients this was maintained for >12 weeks. There was no evidence of DNA interstrand cross-linking in human blood lymphocytes with the use of the comet assay. Evidence of DNA interaction in lymphocytes and tumor cells was shown through a sensitive gamma-H2AX assay. SJG-136 had linear pharmacokinetics across the dose range tested. CONCLUSIONS: SJG-136 was associated with dose-limiting VLS and hepatotoxicity when administered by short injection every 21 days. DNA damage was noted, at all dose levels studied, in circulating lymphocytes. The etiology of the observed toxicities is unclear and is the subject of further preclinical research. Alternative clinical dosing strategies are being evaluated.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease which results from the degeneration of upper and lower motor neurons in the brainstem, spinal cord and motor cortex. Tragically there is no treatment to prevent ALS. The drug Riluzole acts to delay progression, but only by a month or so in this disease that has a survival length of three to five years. The identification of genes that are mutated in patients with ALS would help devise novel therapeutic strategies as much remains to be discovered about the genetics of ALS. Familial forms of the disease account for only 5-10% of patients. Among these familial cases, about 15-20% are caused by mutations in the zinc/copper superoxide dismutase gene, but the genetic basis of the remaining familial cases and the many sporadic cases continues to be largely unknown. / Altogether, the results presented in this thesis came from the use of several strategies to establish the genetic cause of ALS and the related motor neuron disorders like hereditary spastic paraplegia (HSP) and primary lateral sclerosis (PLS). A concerted and collaborative effort was put forth to identify the gene causative for ALS3 on chromosome 18. In addition, a recently reported locus has been confirmed on chromosome 9p for patients that present both ALS and frontotemporal dementia. The major finding involves the discovery of eight mutations in the TARDBP gene in nine patients with sporadic and familial ALS. Furthermore, a large association study evaluated the role of common polymorphisms in the paraoxonase gene cluster in susceptibility to the development of ALS. In the analysis of upper motor neuron diseases, mutations in a novel gene, KIAA0196, were identified for the HSP locus SPG8 on chromosome 8. Finally, the first locus for PLS was discovered on the p-arm of chromosome 4 following genome scan analysis of a large Quebec family with PLS. / These genetic discoveries all contributed novel advances to the field of motor neuron disorders. As more is elucidated regarding the biochemical function of these the proteins encoded by these genes, a more comprehensive picture of ALS and other motor neuron disorders will hopefully emerge.

Thesis (M.S.)--State University of New York at Binghamton, Department of Biological Sciences, 2007. / Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 136-148).

Diss. (sammanfattning) Linköping : Linköpings universitet, 2008. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 106-118).

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 134-155).