No description available.

No description available.

No description available.

No description available.

Cyclic nucleotide-gated (CNG) ion channels are Ca2+ permeable nonselective cation channels that are directly gated by binding of cAMP or cGMP, thus providing a linkage between two important signal molecules, cyclic nucleotides and calcium. They are known to play an important role in sensory transduction and in second-messenger modulation of synaptic neurotransmitter release. Previous studies showed that besides in neuronal cells, CNG were found also in non-neuronal tissues including heart, kidney, blood vessels and spleen, they are reported to be involved in a variety of cell function. / Ion channels play an indispensable role in endothelial cells, which is a unique signal-transducting surface in the vascular system that is responsible in altering vascular tone. The present study investigated the expression and functional roles of the cyclic nucleotide gated channels (CNG) in regulating the intracellular calcium level of vascular endothelial cells using molecular and calcium measurement techniques. / The present study provided evidence that the CNG channels, especially that of CNGA2, were expressed in vascular tissues. I used a variety of different methods, including RT-PCR, northern blot, in-situ hybridization, immunohistochemistry and western blot to study the localization of CNGA2 channels. RT-PCR amplify a CNGA2 fragment of 582bp from RNAs isolated from bovine vascular endothelial cell line, rat vascular smooth muscle cell line and rat aorta. Northern blot analysis detected a 2.3-kilobase (kb) CNGA2 transcript in rat aorta mRNA. The cellular distribution of CNGA2 was further studied by in-situ hybridization, which demonstrated expression of CNGA2 mRNA in human vascular endothelial and vascular smooth muscle cells. Immunohistochemistry data also agreed with those generated from in-situ hybridization. Western blot data also demonstrated proteins of CNG2 was expressed in both human vascular endothelial cells and vascular smooth cells layer. Subcellular localization of CNGA2 inside the vascular endothelial cells was also investigated with the use of a GFP linked CNGA2 channel gene. Taken together, the results showed that CNGA2 proteins were expressed on the plasma membrane of the vascular endothelial cells. (Abstract shortened by UMI.) / Cheng Kwong Tai Oscar. / "July 2005." / Adviser: Xiaoqiang Yao. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3531. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 216-243). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

acase@tulane.edu / Geomorphic features such as fluvial channels and shorelines can act as tape recorders of accumulated tectonic deformation. Because these features can survive in a landscape for up to105 years, the deformation represents tectonic activity over timescales longer than earthquake cycles but shorter than geological timescales. Deformed landscape features can be used to understand the impact of changing tectonic rates on landscape evolution (given information on the tectonic processes involved). Conversely, we can take advantage of how a landscape is expected to evolve and utilize those deviations to explore details of tectonic processes that do not manifest over short timescales (i.e. single earthquakes). Fault slip rate is expected to increase within the overlapping region of two en echelon normal faults, but how increasing slip rate affects the landscape is poorly understood (as discussed in Chapter 1). Additionally, details of this tectonic process that occur over geomorphic timescales are not clearly understood. Chapter 2 of this dissertation explores the impact of fault slip rate increase on fluvial channels during normal fault interaction and linkage. Results of this work show that the landscape responds by increasing channel slope and decreasing channel width before fault segments link. Channel width only shows sustained decreases when a threshold channel slope of about 0.05 is exceeded. In Chapter 3 vertically deformed lacustrine shorelines are mapped along linked faults through the former overlap zones. These results show that despite the presence of linking structures between faults, portions of the former overlapping tips remain active post-linkage for 104 years. Chapter 4 investigates the effect of fault length, spacing, and overlap on the area of relay ramps that drains parallel to fault strike. Twenty-seven sites are examined and results show that for fault lengths below 15 km most of the relay ramp area drains parallel to fault strike, whereas fault lengths >15 km no particular drainage geometry is favored. Data on the overlap/spacing ratio are biased to <2 for faults above ~15 km length. This bias is an inherent characteristic because faults that define low overlap/spacing ratio relays do not link rapidly and are, therefore, preserved within the landscape along large mature fault systems. The results of this dissertation show that, while faults are mechanically interacting, relay ramps are dynamic features that have significant impacts on landscape evolution. Following complete linkage between segments, the relays do not behave as passive structures and can actively deform over significant (>104 years) timescales. Finally, the structural geometry of relay ramps impacts long-term morphodynamics and channel network topology, which directly influences basin sedimentary architecture in extensional basins. / 1 / Michael C. Hopkins

No description available.

Monocytes/macrophages play very important roles in innate and adaptive immunity. Ion channels are small molecules embedded in the cell membrane and they play fundamental roles in cell function. Both topics have been extensively studied in isolation, however the role of ion channels in macrophage function is far less understood. In this thesis, the functional and molecular aspects of two ion channels expressed in macrophages, Kor, a potassium channel, and CLIC1, a chloride channel were studied. The biological function of Kor and CLIC1 in activated human macrophages was examined using ion channel blockers. In addition, the role of CLIC1 in the cell cycling of CHO-K1 cells was also investigated. The in vitro studies showed that Kor and CLIC1 are involved in cytokine production by PMA-activated human macrophages and that CLIC1 is also involved in the cell cycling of CHO-K1 cells. Despite providing interesting data, the results of the in vitro studies were difficult to interpret due to the uncertain specificities of the Cl- channel blockers. Therefore, to understand the biological role of CLIC1 in vivo, a gene targeting experiment was performed to create a CLIC1 knock-out (KO) mouse. This involved cloning the mouse CLIC1 gene, making a targeting vector, producing targeted ES cells, and generating a CLIC1 knock-in (KI) mouse which carries a flag tag at the N-terminal and three loxP sites in the targeted locus. Crossing the CLIC1 KI mouse with the TNAP-Cre mouse, a strain over expressing Cre recombinase under a TNAP promoter, a CLIC1 KO mouse was generated. The initial phenotype analysis showed no major development or growth abnormality in the CLIC1 KO mouse. Instead, hyperplasia of megakaryocytes and possible erythroid cells in the spleen and bone marrow was observed suggesting some degree of abnormality in the haematopoeitic system. Furthermore, a comparison of wild type mice with the CLIC1 KO mouse showed that CLIC1 protein expresses at high levels in monocytes, lymphocytes, platelets, and tissue macrophages of normal animals tissues, such as spleen, kidney (mesangial cell), and liver (kupffer cells). This further indicates that CLIC1 may play a significant role in regulating functions of platelets, lymphocytes, and specially tissue macrophages. More extensive studies can now be performed on the CLIC1 KO mouse to clarify the biological function of CLIC1. In summary, the generation of the CLIC1 KO mouse provides a valuable model to study the biological function of CLIC1 both in vivo and in vitro.

The epithelial sodium channel (ENaC) is a key regulator of salt homeostasis. The classic ENaC consists of three subunits: α, β and γ, which are highly expressed in the kidney and colon where they mediate electrogenic Na⁺ influx into cells under the tight hormonal regulation of aldosterone. A fourth ENaC subunit named [delta]ENaC also generates Na⁺ influx with the β- and γENaC subunits in Xenopus oocytes. However [delta]ENaC differs to the other subunits in its channel properties and tissue distribution, suggesting that [delta]ENaC may possess a physiological role other than salt regulation. A copper-toxicosis related protein called COMMD1/Murr1 was previously identified to directly interact with [delta]ENaC and downregulate [delta]ENaC activity. COMMD1 is linked with multiple ubiquitination pathways, therefore we hypothesised that COMMD1 directly interacts with [delta]ENaC through novel protein-protein interaction motifs and promotes internalisation of [delta]ENaC from the cell surface through enhanced ubiquitination. With the use of GST pulldown assays and coimmunoprecipitation, it was found that the binding of COMMD1 to [delta]ENaC is mediated by the COMM domain of COMMD1, primarily through amino acids 120-150 of COMMD1. Immunocytochemical studies showed that the intracellular interaction between [delta]ENaC and COMMD1 predominantly occurred in the early and recycling endosomes, suggesting that COMMD1 may promote the retrieval of [delta]ENaC from the cell surface to the intracellular pool. COMMD1 mediated a decrease in the [delta]ENaC cell surface population, as shown by a biotinylation surface labelling assay. This may be driven by an ubiquitin-regulated endocytosis, as COMMD1 increased ubiquitination, but not proteasomal/lysosomal degradation, of [delta]ENaC. COMMD1 may promote [delta]ENaC ubiquitination through the action of the ubiquitin ligase Nedd4-2 as coexpression with Nedd4-2 enhanced the COMMD1-mediated decrease in surface [delta]ENaC expression. This is abolished by the addition of the Nedd4-2 downregulator kinase sgk1, suggesting that COMMD1 may downregulate [delta]ENaC through the Nedd4-2/sgk1 pathway. Surface levels of [delta]ENaC may also be affected by XIAP, a RING domain ubiquitin ligase which is able to decrease the levels of COMMD1. Coimmunoprecipitation of endogenous [delta]ENaC and COMMD1 proteins, and the enhanced colocalisation of endogenous [delta]ENaC in the recycling endosomes with transfected COMMD1, indicate that interaction between transfected [delta]ENaC and COMMD1 reflect the intracellular interactions of the endogenous proteins. Taken together, these findings suggest that COMMD1 downregulates [delta]ENaC activity by promoting the internalisation of surface [delta]ENaC into early and recycling endosomes and this may be mediated by enhanced [delta]ENaC ubiquitination via the ubiquitin ligase Nedd4-2.

Thesis (M.S.)--University of Delaware, 2007. / Principal faculty advisor: Robert A. Sikes, Dept. of Biological Sciences. Includes bibliographical references.