<p> IQGAP1 interacts with numerous binding partners through a calponin homology domain (CHD), a WW motif, IQ repeats, a Ras GAP related domain (GRD), and a conserved C-terminal (CT) domain. Among various biological and cellular functions, IQGAP1 plays a role in cell-matrix interactions and actin cytoskeleton dynamics in membrane ruffling and lamellipodium protrusion. Phosphorylation in the CT domain regulates intramolecular interaction and IQGAP1 cellular activity. In a recent study, we discovered that IQGAP1 surprisingly localizes to actively retracting edges, instead of protruding areas, in B16F10 mouse melanoma cells and some other cells types. In these current studies we examined localization of IQGAP1 mutants to retracting versus protruding areas in phorbol ester-stimulated B16F10 cells. Cells were co-transfected with GFP-IQGAP1 full length (GFP-IQGAP1-FL), as an internal control, and one of five Myc-tagged IQGAP1 constructs (FL, CA, &Delta;CHD, &Delta;GRD or &Delta;CT). The cotransfected cells were plated onto laminin for 30 minutes, stained with anti-Myc and anti-WAVE2 antibodies, and normalized fluorescence measurements were made in retracting and protruding areas. Retracting cell areas were defined as GFP-IQGAP1-FL positive and WAVE2 negative, while protruding cell areas were defined as GFP-IQGAP1-FL negative and WAVE2 positive. In retracting areas there were large decreases in both &Delta;GRD and &Delta;CT localization, a slight decrease in &Delta;CHD localization, and normal localization of the CA mutant. In areas of cell protrusion there were large increases in both &Delta;GRD and &Delta;CT localization, and normal localization of &Delta;CHD and CA mutants. These results indicate that two domains, GRD and CT, are essential for normal localization of IQGAP1 to retracting cell areas. Furthermore, our results suggest a model in which IQGAP1 in the areas of cell retraction is in the open, phosphorylated, conformation. Additionally we investigated the knockdown of IQGAP1 in B16F10 cells by means of actin images. Cells were exposed to the lentil virus which contained short hairpin Ribonucleic acid (shRNA) that would silence IQGAP1. Two controls were used in the experiment, untransfected B16F10 cells and B16F10 cells which were exposed to the lentil virus without any shRNA. We found that the knockdown cells were in general much more compact and that they did not polarize. Surface stiffness was investigated in the effect it would have on B16F10 cells. Polyacrylamide gels were made and cross-linked using sulfo-SANPAH. Laminin was added to the cross-linked gels and B16F10 cells were placed on top of the laminin coated hydrogels. Investigation of the cells by means of actin images revealed that surface stiffness had an effect on cell morphology. The 1 kPa surfaces did not allow for spreading of the cells, while the surfaces greater than 100 kPa exhibited normal cell behavior.</p>

<p> Asthmatic human airway smooth muscle cells (hASMCs) exhibit enhanced expression of numerous cytokine-responsive genes but this trend has not been observed for cyclooxygenase-2 (COX-2) expression despite knowledge that conserved regulatory mechanisms exist for cytokine-responsive gene expression. Enhanced expression of cytokine-responsive genes in asthmatic hASMCs has been attributed to differences in histone post-translational modifications and microRNA (miR or miRNA) expression. COX-2 expression is of interest because it serves as a model cytokine-responsive gene and is regulated by epigenetic mechanisms. In other cell types, miR-146a represses COX-2 and Interleukin (IL)-1&beta; expression, directly and indirectly, respectively. Due to sequence homology, miR-146b is predicted to repress the expression of COX-2 and IL-1&beta;. I investigated COX-2 expression in asthmatic and non-asthmatic hASMCS treated with cytomix (IL-1&beta;, tumor necrosis factor (TNF)-&alpha;, and interferon (IFN)-&gamma;). Also, I chose to compare histone acetylation, transcription factor binding, and miR-146a/b expression in asthmatic and non-asthmatic hASMCs to identify any correlations with COX-2 expression. A major goal of this project was to help identify new treatment targets for asthma therapeutics . I hypothesized that asthmatic hASMCs treated with cytomix express more COX-2 and secrete more prostaglandinE2 (PGE2) than non-asthmatic hASMCs due to differences in COX-2 epigenetic regulation. It is reported here that asthmatic hASMCs treated with cytomix expressed more COX-2 (mRNA/protein), and secreted more PGE2 than non-asthmatic hASMCs. Histone H3/H4 pan-acetylation at the COX-2 promoter did not increase with cytomix treatment and was not different in asthmatic and non-asthmatic hASMCs. Treatment of hASMCs with cytomix increased RNA Polymerase II and nuclear factor-&kappa;B binding at the COX-2 promoter with no difference between asthmatic and non-asthmatic hASMCs. Treatment of hASMCs with cytomix increased miR-146a and miR-146b expression with greater miR-146a expression in asthmatic. MiR-146a/b expression in asthmatic hASMCs treated with cytomix did not negatively correlate with COX-2 expression. These results led me to investigate whether miR-146a/b were capable of negatively regulating COX-2 and IL-1&beta; expression in hASMCs. MiR-146a and miR-146b mimics reduced COX-2 and IL-1&beta; mRNA/protein, and PGE2 secretion in hASMCs. MiR-146a and miR-146a/b combination inhibition increased COX-2 and pro-IL-1&beta; protein in hASMCs but not miR-146b inhibition alone. In conclusion, elevated miR-146a expression and histone acetylation are not responsible for increased COX-2 expression in asthmatic hASMCs. MiR-146a is a minor negative regulator of COX-2 and IL-1&beta; expression in hASMCs at physiological expression levels but mimics are capable of antagonizing cytokine-responsive gene expression profoundly. These results coupled with other evidence from the literature indicate that miR-146a/b should be investigated in animal models of asthma to determine if they are relevant asthma drug target in patients that do not respond to current anti-inflammatory therapies.</p>

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2007. / Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5744. Adviser: Mark Estelle. Title from dissertation home page (viewed May 9, 2008).

Thesis (Ph.D.)--University of California, San Francisco, 2005. / Source: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6537. Adviser: Frank McCormick.

Thesis (Ph. D.)--University of California, San Francisco, 2008. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0782. Adviser: Jonathan S. Weissman.

Thesis (Ph. D.)--University of California, San Francisco, 2008. / Source: Dissertation Abstracts International, Volume: 69-06, Section: B, page: 3569. Adviser: Deepak Srivastava.

Thesis (Ph.D.)--University of California, San Francisco, 2006. / Source: Dissertation Abstracts International, Volume: 67-05, Section: B, page: 2370. Adviser: Donald E. Ganem.

Thesis (Ph. D.)--University of California, San Francisco, 2006. / Source: Dissertation Abstracts International, Volume: 68-01, Section: B, page: 0042. Adviser: Kevan M. Shokat.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007. / Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7128. Adviser: Benita Katzenellenbogen. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2008. / Title from PDF t.p. (viewed on Jul 23, 2009). Source: Dissertation Abstracts International, Volume: 69-10, Section: B, page: 5924. Adviser: Christine Quirk.