[Truncated abstract] Androgens inhibit the growth of human breast tumours and have been successfully used to treat breast cancer in women. Expression of the androgen receptor (AR), which mediates androgen action, is upregulated in breast cancer cells and the AR is the most frequently expressed steroid hormone receptor in breast tumours. AR levels and activity are modulated by the activity of other signalling pathways, however interactions between the AR and signalling pathways and the consequent alterations to the androgen responsiveness of breast cancer cells are largely uncharacterised. The extracellular signal regulated kinase (ERK1/2) pathway is hyperactivated in ~30% of breast tumours and these tumours are often associated with low oestrogen receptor-a (ERa) levels, reduced responsiveness to antioestrogen therapies and an overall poorer prognosis. In this thesis, the MCF-7 human breast cancer cell line which expresses ERa, progesterone receptor (PR) and the AR, was used to investigate ERK1/2-mediated regulation of the AR and the androgen responsiveness of cells. Inhibition of ERK1/2 signalling was achieved by treatment of cells with U0126, an inhibitor of MEK1/2, the upstream activator of ERK1/2. Hyperactivation of ERK1/2 signalling was achieved by stably transfecting cells with a plasmid encoding a constitutively active form of the MEK1 protein (¿MEK1), resulting in the isolation of two clonal cell populations stably expressing ¿MEK1, ¿C3 and ¿6B, and a monoclonal cell line stably expressing the empty vector, MT3-1. Steady state AR mRNA levels, quantitated using real-time RT-PCR, were increased following U0126 treatment of MCF-7, MT3-1 and ¿6B cells. Conversely, treatment of cells with 10-8M 5a-dihydrotestosterone (DHT) for up to 72 hours decreased AR mRNA levels, indicating that ERK1/2 hyperactivation did not alter the androgenresponsiveness of AR mRNA. '...' Overall levels of AR phosphorylation were enhanced in ¿6B cells in the absence and presence of ligand, indicating that ERK1/2 hyperactivation either directly or indirectly induced receptor phosphorylation. The AR is localised in the cytoplasm in the absence of ligand and was more rapidly translocated to the nucleus in the presence of DHT in ¿C3 cells, an effect that was abrogated in the presence of U0126, thereby indicating an ERK1/2-specific mechanism. AR transcriptional activity, measured using androgen responsive reporter plasmids was not significantly altered in ¿6B cells in either the absence or presence of DHT, although the trend towards enhanced AR activity may be confirmed in future studies using optimised reporter assays. Consistent with the cell cycle regulatory functions of ERK1/2 signalling, proliferation of ¿C3 cells and ¿6B cells was increased in comparison to that of MT3-1 and MCF-7 cells. Treatment of ¿C3 cells and MCF-7 cells with 10-10 – 10-8M DHT produced similar inhibition of proliferation (~40%) during 8 days of culture, with no evidence of cytotoxicity. The results obtained in this thesis demonstrate that while ERK1/2 signalling regulates AR phosphorylation, processing and intracellular localisation, ERK1/2 hyperactivation in breast cancer cells does not inhibit the anti-proliferative effects of androgens. These findings support the development of tissue-specific androgenic treatments for breast tumours including poor prognosis tumours exhibiting ERK1/2 hyperactivation.

The thesis begins with an introduction to signal transduction and an analysis of current understanding of plant signal transduction. There are similarities between plants and animals, but also key differences, including lack of protein kinase C and of a cAMP signaling pathway in plants, and presence in plants of calcium dependent protein kinase (CDPK), which has a kinase catalytic domain contiguous with a C-terminal calmodulin-like domain. The next section examines protein kinase activity in the plasma membrane (PM) of zucchini hypocotyls. Zucchini PM contains four or more polypeptides with calcium-requiring protein kinase activity. The enzymes appear to be tightly associated with the PM, and at least three are recognized by monoclonal antibody to soybean soluble CDPK. Total proteins from several different organs of zucchini seedlings contain kinases with molecular weights similar to the hypocotyl PM enzymes. In the third section details of partial purification of the solubilized PM kinases are presented. Kinases which do not crossreact with anti-CDPK monoclonal antibody were resolved by anion exchange from ones which do crossreact. Peptide mapping was used to test the relationship between the kinases. Results of peptide mapping suggest that at least three types of protein kinase are present in zucchini PM, two of which are immunologically similar to CDPK and one of which is not. The last section concerns the potential for testing interactions between PM protein kinases and plasma membrane auxin binding proteins (ABP's) by use of photoaffinity labeling of ABP's. Causes of variable photoaffinity labeling by an azido-IAA are considered. Labeling of both the tomato mutant diageotropica and the parent VFN membranes was inexplicably inconsistent. / Graduation date: 1994

Thesis (M.S. in Cancer Biology)--Vanderbilt University, May 2007. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 160-193).

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 130-153).

IDENTIFICATION OF PHOSPHORYLASE KINASE ALPHA SUBUNIT BINDING PARTNERS IN SKELETAL MUSCLE Soleil Archila August 2004 67 Pages Directed by: Nancy A. Rice, Sigrid Jacobshagen, and Claire A. Rinehart Department of Biology Western Kentucky University Phosphorylase kinase (PhK) integrates neural, hormonal, and metabolic signals in skeletal muscle to tightly regulate glycogen breakdown and energy production. Structurally, PhK is among the largest and most complex kinases known with a stoichiometry of (αβγδ)4 and a mass of 1.3x106 Da. The catalytic γ subunit is allosterically controlled through alterations in quaternary structure initiated by the regulatory α, β and δ subunits. In this study we have chosen to examine the largest regulatory subunit, α. In addition to participating in intramolecular interactions within PhK, α is hypothesized to associate extrinsically with skeletal muscle proteins due to its peripheral location in the holoenzyme. To identify potential muscle protein interactors, the C-terminus of α , amino acid residues 1060-1237, was screened against a rabbit skeletal muscle cDNA library via a yeast two-hybrid assay. Interactions were selected by auxotrophic growth of yeast in the absence of leucine and by expression of β-galactosidase activity. Thirtyseven potential positive clones were initially observed. Secondary selections resulted in 13 putative clones being isolated of which 8 were identified as thyroid hormone receptor interacting protein 10 (TRIP10). TRIP10, also known as Cdc42 interacting protein 4 (CIP4), is highly expressed in skeletal muscle and is thought to act as a regulator of the actin cytoskeleton. The remaining 5 clones were identified as tetratricopeptide repeat protein 1 (TPR1). TPR1 is an adaptor protein that functions in a wide array of cellular processes and is considered a highly specific adaptor. The results reported herein are intriguing given that PhK is associated with the sarcoplasmic reticulum in the A-band / actin filament overlap zone and has previously been shown to interact with the thin filament protein nebulin. To our knowledge, this is the first report to identify TRIP10 as a potential sarcomeric protein, and these findings further suggest a role for PhK in muscle assembly and architecture.

Thesis (Ph. D.)--University of Oregon, 2001. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 163-175). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 181-193). Also available on the Internet.

Thesis (M.S.)--The University of North Carolina at Greensboro, 2008. / Directed by Paul Steimle; submitted to the Dept. of Biology. Title from PDF t.p. (viewed Mar. 19, 2010). Includes bibliographical references (p. 30-31).

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 109-124).