Rho GTPases are critical regulators of actin cytoskeletal dynamics. The three most well characterized Rho GTPases, Rac1, RhoA and Cdc42 share a common inhibitor, RhoGDI. It is only recently becoming clear how upstream signals cause the selective release of individual Rho GTPases from RhoGDI. For example, our laboratory showed that diacylglycerol kinase zeta (DGKz), which converts diacylglycerol (DAG) to phosphatidic acid (PA), activates PAK1-mediated RhoGDI phosphorylation on Ser-101/174, causing selective Rac1 release and activation. Phosphorylation of RhoGDI on Ser-34 by PKCa has recently been demonstrated to selectively release RhoA, promoting RhoA activation. Here, I show DGKz is required for optimal RhoA activation and RhoGDI Ser-34 phosphorylation. Both were substantially reduced in DGKz-null fibroblasts and occurred independently of DGKz activity, but required a function DGKz PDZ-binding motif. In contrast, Rac1 activation required DGKz-derived PA, but not PDZ-interactions, indicating DGKz regulates these Rho GTPases by two distinct regulatory complexes. Interestingly, RhoA bound directly to the DGKz C1A domain, the same region known to bind Rac1. By direct interactions with RhoA and PKCa, DGKz was required for the efficient co-precipitation of these proteins, suggesting it is important to assemble a signalling complex that functions as a RhoA-specific RhoGDI dissociation complex. Consequently, cells lacking DGKz exhibited decreased RhoA signalling downstream and disrupted stress fibers. Moreover, DGKz loss resulted in decreased stress fiber formation following the expression of a constitutively active RhoA mutant, suggesting it is also important for RhoA function following activation. This is consistent with the ability of DGKz to bind both active and inactive RhoA conformations. Collectively, these findings suggest DGKz is central to two distinct Rho GTPase activation complexes, each having different requirements for DGKz activity and PDZ interactions, and might regulate the balance of Rac1 and RhoA activity during dynamic changes to the actin cytoskeleton.

Differentiation is the process of regulated gene expression that gives rise to different phenotypes from a common genotype. Skeletal muscle differentiation, myogenesis, is a good example of this process. Skeletal muscle is susceptible to injury due to direct or indirect causes. If left unrepaired, these injuries may lead to a loss of muscle mass, locomotive deficiency, and even lethality. Thus, understanding the molecular mechanisms behind this process is an important first step in the design of treatment for muscle-related diseases. Once myogenesis is induced, the expression of MRF proteins, such as MyoD and myogenin, is maintained at high levels in myofibers without the need to increase their rates of transcription, suggesting a role for post-transcriptional regulatory mechanisms. HuR is a ubiquitously expressed member of the embryonic lethal, abnormal vision (ELAV) family of RNA binding proteins that is known to post-transcriptionally regulate its target messages. Here, I demonstrate that, in the C2C12 muscle cell line, HuR is a required factor for both the initiation and maintenance of the myogenic process. First in vitro RNA Electro-Mobility Shift Assays (REMSA) and immunoprecipitation experiments demonstrated that HuR specifically binds to the AU-rich elements (AREs) that are present in the 3' untranslated regions (3'UTRs) of the MyoD and myogenin mRNAs. In the absence of HuR at the time of differentiation induction, accomplished using the siRNA technology, the expression of the MyoD and myogenin messages is significantly reduced, leading to inhibition of myogenesis. At this early stage in the differentiation process, HuR, a shuttling protein, is predominantly nuclear; localization that is mediated by the import receptor Transportin2 (Trn2). Nuclear HuR was determined to be required for the negative regulation of nucleophosmin (NPM) translation. Forced overexpression of NPM, resulting in differentiation inhibition, shows that its downregulation is a requirement for induction of the differentiation process. Late in myogenesis, however, NPM RNA is no longer expressed, and HuR is seen to accumulate in the cytoplasm of myotubes. This cytoplasmic accumulation results from dissociation of HuR from Trn2 due to caspase-dependent cleavage within its HNS region. Specifically blocking HuR import through the use of cell-permeable peptides, as well as RNAi-mediated depletion of Trn2, leads to an increase in cytoplasmic HuR, as well as increased cytoplasmic localization and stabilization of the MyoD and myogenin messages, and a corresponding enhancement of differentiation. Overall, we conclude that HuR is required for myogenesis due to its ability to post-transcriptionally regulate genes required for the process, and that HuR itself is regulated at the level of its subcellular localization, mediated by the import receptor TRN2.

Myocardial infarction activates the renin-angiotensin system, consequently upregulating aldosterone production that may stimulate pathological cardiac fibrosis via mineralocorticoid receptor (MR) activation. Results presented in this thesis were derived from an in vitro experimental model using cultures of human cardiac fibroblasts to study the effect of aldosterone on elastin production. They first confirmed that treatment with 1-50 nM of aldosterone leads to a significant increase in collagen type I production via MR activation. Most importantly, we discovered that treatment with 1-50 nM of aldosterone also increases elastin mRNA levels, tropoelastin synthesis, and elastic fiber deposition. Strikingly, pretreatment with MR antagonist spironolactone did not eliminate aldosterone-induced increases in elastin production. Interestingly, while cultures treated with elevated aldosterone concentrations (100 nM and 1 µM) showed a further increase (~3.5-fold) in collagen and (~3-fold) in elastin mRNA levels, they demonstrated subsequent increases only in the net deposition of collagen but not elastin. In fact, cultures treated with elevated aldosterone concentrations displayed a striking decrease in the net deposition of insoluble elastin, which could be reversed with spironolactone or with MMP inhibitors doxycycline or GM6001. Most importantly, we discovered that the pro-elastogenic effect of aldosterone involves a rapid increase in tyrosine phosphorylation of the insulin-like growth factor-I receptor (IGF-IR) and that the IGF-IR kinase inhibitor AG1024 or an anti-IGF-IR neutralizing antibody inhibits both IGF-I- and aldosterone-induced elastogenesis (Bunda et al., Am J Pathol. 171:809-819, 2007). Furthermore, we showed that the PI3 kinase signaling pathway propagates the elastogenic signal following IGF-IR activation and that activation of c-Src is an important prerequisite for aldosterone-dependent facilitation of the IGF-IR/PI3 kinase signaling. Results of explorative microarray analysis of 1 hour aldosterone-treated cultures revealed that aldosterone treatment upregulated expression of a heterotrimeric G protein, Gα13, that activates the PI3 kinase signaling pathway. We additionally demonstrated that aldosterone treatment transiently increases the interaction between Gα13 and c-Src and that siRNA-dependent elimination of Gα13 inhibited the pro-elastogenic effect of aldosterone. In summary, aldosterone, which stimulates collagen production in cardiac fibroblasts through the MR-dependent pathway, also increases elastogenesis via a parallel MR-independent pathway involving the activation of Gα13, c-Src, and IGF-IR/PI3 kinase signaling.

Hornification is the term used to describe the irreversible changes that occur in cellulose fibers due to recycling. While the effects of hornification have been documented, there are several conflicting hypotheses that attempt to explain the causes. In this research, AFM surface adhesion measurements made on virgin and recycled bleached kraft pulp show that recycling increases the apparent hydrophilicity of the fiber surface. Yet, the water retention values and tensile strength decrease as expected, which is consistent with internal cross-linking of the bonding sites and a reduction in hydrophilicity. Recycling does not affect the amount of monolayer water bound to the fiber surface indicating that the pore water is reduced but not the water bound to fiber surfaces. It is proposed that the contact area between the AFM tip and the fiber is greater for recycled material than for virgin. Image analysis of the fiber surface supports this conclusion, revealing a decrease in the surface roughness. The irregularity of fiber surfaces is apparent in AFM images at all but the smallest scales, with macroscopic fiber features dominating the roughness analyses of images larger than 2500 square nanometers. Hence, in this instance, the surface adhesion values are more a measure of the topography of the surface than of its chemistry. An application to newsprint is illustrated, revealing the limitations of AFM analysis of samples with a high degree of variability.

The axilla is a major source of human body odour from which the characteristic musky, urinous or acidic odours emanate, and are predominantly due to bacterial metabolism of the protein-rich fluid secreted by the apocrine and sebaceous glands located in this area (Senol and Fireman, 1999). Clothing has been implicated in contributing to body odour intensity, possibly even increasing the intensity (Dravnieks, et al., 1968; Shelley, et al., 1953) by the transfer of secretions, skin debris and bacteria from the body to the fabric substrate. Despite much anecdotal evidence indicating that some fibres and fabrics are better at limiting odour intensity than others, there appears to be no published research confirming this. The purpose of this study therefore, was to determine whether fabrics varying in fibre content (cotton, wool, polyester) and fabric knit structure (interlock, single jersey, 1x1 rib) differed in the extent to which they retained and emanated axillary odour following wear, and whether the intensity of odour was linked to the number of bacteria transferred to the fabrics. A procedure for collecting odour on fabrics was developed as was a method for evaluating odour through use of a sensory panel. Total aerobic bacteria and aerobic coryneform bacteria extracted from the fabrics were counted to determine if an association between bacterial counts and fabrics existed. Sensory analysis recognises the unique capability of humans as odour-detecting instruments whereas, instrumental analysis has the potential to offer information on the concentration and identification of axillary compounds, which a human assessor cannot. To investigate a new method for detecting axillary odour on apparel fabrics, proton transfer reaction mass spectrometry (PTR-MS) was used to analyse volatiles emitted from fabrics differing in fibre type. After removal of garments from the human body, axillary odour can be detected on fabrics, with the intensity of odour being strongly influenced by the fibre type from which the fabrics had been made. Polyester fabrics emanated odour of high intensity, cotton that of mid-low odour intensity, and wool fabrics were low odour. Fabric structural properties such as thickness, mass per unit area and openness of knit structure also had an effect on odour intensity. However, as the principal factor influencing odour intensity was fibre, only fabrics characterised by a high intensity (i.e. polyester) were influenced by structural properties. Differences in odour intensity among fabrics were not necessarily related to bacterial numbers, and no �inherent antimicrobial� properties were evident for any of the fabrics. Bacterial populations persisted in all fabrics up to 28 days. A decline in numbers was apparent for high-odour polyester fabrics, while numbers in low-odour wool fabrics remained relatively stable. PTR-MS detected compounds likely to be short-chain carboxylic acids which increased in the headspace above the polyester fabrics after 7 days. However, this increase was not evident for either the wool or cotton fabrics. Therefore, bacterial numbers per se cannot be a predictor of the odour intensity emanating from fabrics at least on the basis of these fabrics and fibres. The intensity of axillary odour emanating from fabrics was found inversely related to fibre hygroscopicity. Keywords:fibre content, fabric structure, axillary odour, sensory analysis, bacteria, corynebacteria, instrumental analysis, PTR-MS

Thesis (M.S.)--University of Missouri--Rolla, 2007. / Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed March 25, 2008) Includes bibliographical references (p. 52-54).

Thesis (M.S. in Civil Engineering)--Kansas State University, 2007. / Includes bibliographical references (p. 104-106). Also available online.

Thesis (Ph. D.)--Ohio University, November, 2000. / Title from PDF t.p.

Thesis(M.S.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.

Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Sciences, 1990. / Includes bibliographical references (leaves 246-280) and index.