In living systems, ion channels are membrane transport proteins that provide pathways for the passive diffusion of ions through lipid membranes. The flow of ions across membranes is the basis of many important physiological processes, including but not limited to the regulation of membrane potential, transepithelial transport and cell volume. While many efforts have been made to understand the biological roles of natural ion channels, the biological activities of artificial ion channels remain largely unknown. Recently, it was reported that a small molecule 1, which forms synthetic chloride (Cl–) channels in membranes via self-assembly, is capable of modulating vascular functions. In this thesis, novel small molecules that are structurally similar to 1 are shown to form artificial ion channels in membranes. Together with 1, the effects of these small molecules on the contractile activities of smooth muscles and epithelial ion transport are explored. The therapeutic implications of the findings are also discussed. A collection of small molecules was screened using liposome-based fluorescence assays. In these assays, the ability of the synthetic compounds to modulate membrane potential was monitored. The screening yielded compound 3 that formed synthetic potassium (K+) channels in liposomal membranes, although the liposome-based fluorescence experiments suggested that 3 also transported Cl–. Two derivatives of 3, namely, compounds 2 and 4 were also examined. Single-channel recording experiments suggested that 2 forms synthetic Cl– channels in liposomal membranes. The effects of compounds 2 and 3 on the functions of the vascular smooth muscle are explored. Using confocal imaging, it was shown that both 2 and 3 counteracted the effects of high-K+ depolarizing solution on membrane potential and intracellular Ca2+ concentration ([Ca2+]i) in cultured vascular smooth muscle cells. 2 and 3 also relaxed mice aortic rings pre-contracted with high-K+ solution. These observations can be explained in terms of the Cl– transporting functions of 2 and 3. To determine the potential for developing the compounds into bronchodilators, the effects of compounds 1 and 3 on the contractile activities of the airway smooth muscle (ASM) were explored using organ bath technique. The contractile activities of the trachea isolated from Sprague-Dawley (SD) rats were first characterized. Among the contractile agents used, only potassium chloride (KCl), cholinergic agonists, serotonin and endothelin-1 were contractile to the SD rat trachea. 1 and 3 relaxed the ASM pre-contracted with KCl, whereas the contractions induced by other agonists were not affected. The ability of compounds 2, 3 and 4 to modulate ion transport across cultured epithelia was tested by the short-circuit current measurement technique. It was shown that the compounds were capable of inducing Cl– secretion when applied to the apical side of airway and colonic epithelia. Importantly, the synthetic compounds induced apical Cl– secretion in immortalized cystic fibrosis (CF) bronchial epithelia. This suggests that the synthetic compounds may be used to correct the anion transport defect in CF epithelia. In summary, the small-molecule based synthetic ion channels demonstrated two important general functions of natural ion channels, namely, the regulation of membrane potential and epithelial ion transport. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Surgery / Master / Master of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

Epithelial cells lining the upper airways are characterized by low sodium absorption and elevated chloride secretion. Together, the movement of these ions creates the osmotic drive to hydrate the airways. Recent studies indicate that influenza is capable of directly modulating the vectorial transport of sodium and chloride ions. However, the direct impact of influenza has not been studied with respect to potassium channels. This is significant because potassium conductance creates the driving force for chloride secretion. Disruptions to this process leads to edema formation in the lungs and can subsequently cause Acute Respiratory Distress Syndrome. Additionally, it has been demonstrated that the induction of pro-inflammatory cytokines in infected cells may contribute to altered ion channel function, further exacerbating edema formation. The purpose of this study was to assess the direct and indirect effects of influenza virus infection on potassium and chloride ion channel function in a secretory epithelial cell model. In order to assess the direct effects we exposed polarized epithelial cell monolayers to varying doses of H1N1 virus. Potassium and chloride channel function was measured by means of short-circuit current in an Ussing chamber. The immune response to viral infection was determined by RT-qPCR and Bioplex suspension array. Virus conditioned media (CM), and IL-8 were used to characterize the indirect effects on non-infected cells. We observed an increase in chloride secretion, consistent with edema formation, when 60% of the epithelium was infected, and after CM treatment. This observation correlated with increased potassium channel conductance through the calcium-activated (KCNN4) and cAMP-activated potassium channels (KCNQ1), which was ameliorated upon specific inhibition of these channels. The data suggest that the mixture of pro-inflammatory cytokines induced by viral infection directly up-regulate these potassium channels. However, treatment with IL-8 also appears to increase chloride secretion, although the underlying mechanism remains to be determined, as it is not mediated through KCNN4 and KCNQ1. We conclude that the strong induction of cytokines in infected cells act in a paracrine manner on non-infected cells to increase potassium channel conductance. This up-regulation of potassium channels subsequently drives an increase in chloride secretion, leading to fluid build-up in the lungs and edema formation.

This work helps to determine the extent of immune system involvement in the adaptive response to subacute ruminal acidosis (SARA) in three parts. The first (Chapter 2) uses non-lactating cows to study specific changes in inflammatory protein expression in which SARA is created. The second (Chapter 3), uses the same model as Chapter 2. However, in this case, lactating cows are used to help establish the time course for adaptation to acidosis. The third part (Chapter 4) delineates the genomic changes that occur in the rumen epithelium when a therapeutic intervention is introduced using exogenous supplemental butyrate. In the first experiment, the expression of the extracellular matrix (ECM) proteins type IV collagen and laminin β1 decreased, and the monocarboxylate transporter MCT1, increased during the acidotic challenge. Nuclear factor of activated T-cells, NFATc2, and tumour necrosis factor alpha (TNF-α) decreased while interleukin-1 beta (IL-1β) increased during the experimental treatment period. Chapter 3 measured lipopolysaccharide (LPS) and its carrier, LPS binding protein, LBP, which were found to be elevated due to SARA. Moreover, NFATc2 was reduced during this period. Exogenous butyrate resulted in increased plasma LBP, plasma beta hydroxyl butyrate (BHBA), and ruminal butyrate. Milk parameters (total protein and fat) were unaffected by treatment, as were rumen LPS, acetate, valerate, isovalerate, and isobutyrate. Moreover, exogenous butyrate increased gene transcription of genes involved in non-specific host defences (NHSD) such as mucin, and remodelling (RM), such as matrix metallopeptidase 16 (MMP16), and decreased the transcription of genes of the immune response (IR), such as nuclear factor kappa B2 (NFκB2). Together, these three experiments have demonstrated that although wound healing is mediated by the immune system in more severe models of epithelial damage, our model of SARA did not involve full-thickness, penetrating lesions and hence did not involve the systemic immune system to such a degree than was previously thought. In addition, we were able to demonstrate that the addition of butyrate to this model of grain-induced acidosis was beneficial, as it decreased the local inflammatory response and helped the epithelium adapt to its harsher environment. / Agriculture and Agri-Food Canada, the National Sciences and Engineering Council of Canada (NSERC), the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA), the Canadian Dairy Commission (CDC), and the Dairy Farmers of Ontario (DFO).

The vagina is one organ of the body which has not been studied exhaustively. Moreover, most of the studies found in the contemporary literature have been performed on women affected by a variety of genital diseases. In the present study the vaginal epithelium was examined with a histological method, morphometry, whereby cyclical changes related to hormonal variation during the menstrual cycle were demonstrated. Determination of the quantity of estrogen receptors in the vaginal epithelium on two occasions during the menstrual cycle revealed a significantly greater number in the follicular than in the luteal phase. The results of these studies indicate the presence of a menstrual variation in the vaginal epithelium comparable to that in the endometrium. Phenoxymethylpenicillin (pcV) was used as a marker substance to study the dynamics of the transport mechanisms into the vagina. PcV was found to accumulate in the vaginal fluid and high concentrations persisted for a long period of time. In hysterectomized women, the appearance of pcV in the vaginal fluid followed the same pattern. Consequently, the substance is transported through the vaginal wall and need not enter with the secretions from the internal genitalia. The greatest concentration of pcV was in the distal portion of the vagina, possibly due to the specific internal circulation of fluid within the vagina. Bacterial vaginosis as an example of an ‘ecological disease’ has been studied with regard to the formation of endotoxin, a constituent of the cell wall of Gram- negative bacteria. Large amounts of endotoxin were found and the clinical implication of this finding has been pointed out. Furthermore, the influence of pcV on the vaginal microbial flora of healthy women has been investigated. A change from a situation with predominance of lactobacilli to the appearance of Gram-negative rods was observed. In one of the women the lactobacilli disappeared completely and were replaced by E. coliand high levels of endotoxin in the vaginal fluid were found. This study demonstrates the complexity of the ecological balance of the vaginal microbial flora and illustrates the difficulty of defining a ‘normal’ vaginal condition. Is there any unquestionable state of ‘normality’ even in a healthy woman free from symptoms of genital disease? / <p>S. 1-22: sammanfattning, s. 25-64: 6 uppsatser</p> / digitalisering@umu

The role of the retinal pigment epithelial cells on the viability and renewal of photoreceptors has been well demonstrated in the Royal College of Surgeons (RCS) strain of rat. These rats are characterized by an inherited time-dependent degeneration of their photoreceptors. This degeneration is apparently due to the inability of the retinal pigment epithelial cells to adequately ingest fragments of photoreceptor membrane that are shed during the course of photoreceptor membrane renewal. The buildup of photoreceptor material in the interphotoreceptor space ultimately leads to the degeneration of photoreceptors in these animals. With regard to the pigment epithelial cells, neither the mechanism mediating the ingestion process in normal rats nor the nature of the defect of this process in RCS rats is understood.It is the goal of this proposed research to assay for the presence of phospholipase C in retinal pigment epithelial (RPE) cells and to determine possible modulators of the enzyme in an attempt to associate this with the process of phagocytosis. / Department of Biology

The products of the Cdx genes, Cdx1, Cdx2 and Cdx4, are known to play essential roles in many developmental processes including neural tube closure, axial elongation and patterning the anterior-posterior axis of the developing embryo. Cdx1 and Cdx2 are both expressed in the endoderm of the embryo and persist throughout adulthood in the intestinal epithelium, but their functions and mechanisms of action in this lineage are poorly understood, in part due to the peri-implantation lethality of Cdx2-/- mice. To circumvent this limitation, a conditional loss of function strategy was used to inactivate Cdx2 in the intestinal epithelium. These conditional mutants were also crossed to Cdx1-/- mice, which are viable and fertile, to examine potential functional compensation between these family members. The major findings of this study are that Cdx2 regulates patterning and differentiation of the small intestinal epithelium, while Cdx1 does not appear to make a contribution to either process. Furthermore, Cdx operates upstream of Notch ligand Delta-like 1 (Dll1) in endoderm and mesoderm derivatives, demonstrating that Cdx function is similar in different lineages. Finally, Cdx2 cannot fulfill the requirement for Cdx1 in regulation of its own promoter in the intestine. This is the first in vivo evidence that these two family members have context-dependent functional specificity. Altogether, this study underscores critical roles and mechanisms of action for Cdx members in the developing intestine and mesoderm.

Offprints of the author's articles inserted / Bibliography: leaves 230-265 / List of the author's publications: leaves 266-263 [i.e. 267] / 263 [i.e. 268] leaves : ill. (1 col.) ; 31 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Dental Science, 1984

Thesis (Ph. D.)--University of Western Sydney, 2002. / "This thesis is submitted in fulfilment of the requirements of the degree of Doctor of Philosophy to the University of Western Sydney School of Biological Sciences."t.p. Includes bibliographical references (leaves 138-150).