Thesis (Ph. D.)--Institute of Paper Chemistry, 1939. / Includes bibliographical references (leaves 72-73).

Thesis (Ph. D.)--University of Oregon, 2003. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 137-145). Also available for download via the World Wide Web; free to University of Oregon users.

Plasma membrane calcium/calmodulin-dependent calcium ATPases (PMCAs) are high affinity calcium pumps regulating many calcium-dependent processes and advances in its characterisation have discovered that it may play a novel role in signal transduction pathways. It was the aim of this work to further characterise and confirm the role PMCA plays in regulating calcium/calmodulin-dependent signal transduction pathways. PMCA4 has already been shown to inhibit the NFAT family of transcription factors by its interaction with calcineurin A in mammalian cells when ectopically expressed. This prompted the investigation into other isoforms of PMCA that may interact with the calcium/calmodulin-dependent calcineurin, to determine if this interaction was isoform-specific in a variety of cell lines. Endogenous proteins were isolated by immunoprecipitation with calcineurin A antibody and the presence of PMCA isoforms was determined by western blot using isoform-specific antibodies. This work has demonstrated that the PMCA and calcineurin interaction occurs in vitro at endogenous levels in MCF-7 human breast adenocarcinoma cells and endothelial cells and is isoform specific, predominantly for PMCA2. The characterisation of the PMCA2b-calcineurin A interactive domain was performed and it was demonstrated that PMCA2b significantly inhibits the NFAT/calcineurin pathway. These results indicate that PMCA2 is important in regulating the calcineurin/NFAT pathway in tissues where it is highly expressed. This work also demonstrates that the Flag-tagged, characterised interaction domain of PMCA2 with calcineurin, F-PMCA(462-684) when overexpressed, can disrupt the inhibitory PMCA2/calcineurin interaction in endothelial cells and significantly increase calcineurin activity. The expression of PMCA in endothelial cells prompted the investigation of calcium/calmodulin-dependent proteins in endothelial cells as evidence for the important role of PMCA in regulating signal transduction pathways. Nitric oxide synthases have been shown to be regulated by PMCA4 in cardiac cells. To further characterise the regulation of NOS by PMCA, this work shows that there is a novel molecular interaction between endogenous eNOS and the plasma membrane calcium ATPase (PMCA) in HUVEC primary endothelial cells. PMCA2 has been identified as the major isoform interacting with eNOS in endothelial cells. The interaction between the two proteins has been mapped to the region 735-934 of eNOS and 462-684 of human PMCA2b. NO production was found to be inhibited by ectopic expression of PMCA2b in HUVEC cells. Moreover, disruption of the interaction between endogenous PMCA and eNOS by overexpression of theFlag-tagged, PMCA2b interaction domain, F-PMCA2(462-684), significantly increased NO levels in activated HUVEC endothelial cells. In summary, these results offer strong evidence for a novel functional interaction between endogenous PMCA and eNOS in endothelial cells, suggesting a role for endothelial PMCA2 as a negative modulator of eNOS activity, and, therefore, NO-dependent signal transduction pathways. Overall this is a novel discovery which clearly demonstrates that PMCA is an important regulator of calcium/calmodulin-dependent signal transduction pathways in various cell types. Parts of this work have been published; ‘Holton, M., Yang, D., Wang, W., Mohamed, T.M., Neyses, L. and Armesilla, A. (2007) The interaction between endogenous calcineurin and the plasma membrane calcium-dependent ATPase is isoform specific in breast cancer cells. FEBS letter. 581(21), 4115-4119.’ and presented at ‘The 14th congress of calcium binding proteins, La Palma, Canary Islands, Spain. 2007’ and ‘The 25th Conference of the European Society on Microcirculation (August 26-29, 2008, Budapest, Hungary).’

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

This thesis describes novel microel ectromechanical system (MEMS) based electrochemical gas sensors and methods of fabrication. This thesis presents the research in two parts. In the first part, a method to handle a thin silicon wafer using an electrochemically active adhesive is described. Handling of a thin silicon wafer is an important issue in 3D-IC manufacturing where through silicon vias (TSVs) is an enabling technology. Thin silicon wafers are flexible and fragile, therefore difficult to handle. In addressing the need for a reliable solution, a method based on an electrochemically active adhesive was developed. In this method, an electrochemically active adhesive was diluted and spin coated on a 100 mm diameter silicon wafer (carrier wafer) on which another silicon wafer (device wafer) was bonded. Device wafer was subjected to post processing fabrication technique such as wafer thinning. Successful debonding of the device wafer was achieved by applying a voltage between the two wafers. In another part of the research, a fabrication process for developing a functional nanoporous material using atomic layer deposition is presented. In order to realize a nanoporous electrode, a nanoporous anodized aluminum oxide (AAO) substrate was used, which was functionalized with very thin layers (~ 10 nm) of platinum (Pt) and aluminum oxide (Al2O3) using atomic layer deposition. Nanoporous material when used as an electrode delivers high sensitivity due to the inherent high surface area and is potentially applicable in fuel cells and in electrochemical sensing. The second part of the thesis addresses the need for a high performance gas sensor that is applicable for asthma monitoring. Asthma is a disease related to the inflammation in the airways of the lungs and is characterized by the presence of nitric oxide gas in the exhaled breath. The gas concentration of above approximately 50 parts-per-billion indicates a likely presence of asthma. A MEMS based electrochemical gas sensor was successfully designed and developed to meet the stringent requirements needed for asthma detection. Furthermore, to enable a hand held asthma measuring instrument, a miniaturized sensor with integrated electrodes and liquid electrolyte was developed. The electrodes were assembled at a wafer-level to demonstrate the feasibility towards a high volume fabrication of the gas sensors. In addition, the designed amperometric gas sensor was successfully tested for hydrogen sulphide concentration, which is a bio marker for bad breath. / <p>QC 20150907</p>

Male-typical copulatory behaviors such as mounting and intromission are dependent on testicular androgens in most vertebrates, being eliminated by castration and re-instated by administration of exogenous testosterone. Testosterone implants in the preoptic area (POA) can re-instate behavior as effectively as systemic testosterone replacement, implicating this area as a critical locus of hormonal gating. The cellular mechanisms underlying this gating phenomenon are not well understood, but according to one model, testosterone induces an up-regulation of nitric oxide synthase (NOS) in the POA, increasing nitric oxide synthesis following exposure to a sexual stimulus. Nitric oxide in turn, possibly through its effect on catecholamine turnover, influences the way the stimulus is processed and enables the appropriate copulatory behavioral response. The experiments described in this Dissertation were designed to test this model as it pertains to hormonal gating in Cnemidophorus lizards. Specifically, experiments were conducted to test the predictions that nitric oxide synthesis inhibition would suppress the expression of behavior; that preoptic nitric oxide synthesis would be greater in animals expressing copulatory behavior; and that preoptic NOS expression, at both the mRNA and the protein levels, would be greater in animals exposed to testosterone than in animals deprived of hormone. All three of these predictions were upheld, offering support to the model as described. / text

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Electrical and Electronic Engineering / Doctoral / Doctor of Philosophy