This thesis evaluates the utility of a geochemical technique for the investigation of palaeoenvironmental change in south west England. The method, EDMA (Energy Dispersive X-ray Micro Analysis), is a rapid, non-destructive analysis tool, capable of detecting a large range of geochemical elements. This research examines the most appropriate method of sample preparation for organic soils and peats, and investigates the reliability of results gained from EDMA with respect to conventional bulk geochemical techniques. A detailed study focused on a range of different sedimentary sites in south west England where a variety of palaeoenvironmental changes were thought to occur. Pollen analysis was undertaken on the same sedimentary material, and provided complementary information on the nature and scale of vegetation change through time. Sediments from a coastal valley mire near North Sands, Salcombe, revealed information relating to the processes of sea-level change in this part of south Devon and the subsequent autogenic processes as the sediment accumulated through time. A range of sites were located on the granitic upland of Dartmoor. A raised bog, Tor Royal, provided data relating to the changing nature of the central upland landscape from late Mesolithic times to the present day. Two soligenous sites, Upper Merrivale and Piles Copse, sought to investigate the activities of postulated anthropogenic activity at a much smaller spatial scale, with particular interest placed upon the evidence for deforestation activity and the utilisation of the local mineral resources. The last site, Crift Down, a lowland spring fed valley mire utilised geochemical and palynological fluxes within the peat to investigate processes and activities associated with archaeological evidence for Medieval tinworking in this area of Cornwall. The results from the EDMA investigations, and comparable studies using other geochemical methods including EMMA, AAS and flame photometry, suggest the technique to have greatest applicability as a first stage tool in the analysis of general activities of past environmental change. The technique was found to yield reliable results for the major elements (Si, Al, 5, Fe, Ca, K, Na and Mg), but is generally incapable of providing useful data on heavy metal elements. The data from south west England suggest the method to reflect activity at a range of different scales, and as part of a structured programme of analysis may contribute information to allow a more holistic environmental reconstruction to be made.

Thesis (Ph. D.)--Lehigh University, 2005. / Includes vita. Includes bibliographical references (leaves 261-275).

X-ray diffraction techniques were used to determine thicknesses, and strains in sputtered polycrystalline thin films of molybdenum and titanium on silicon single crystal substrates. The thickness determination method used was an extension of a previous method wherein the orientation function determined from experimental data obtained in only a part of the angular range in chi could be fitted with Legendre polynomials and extrapolated to chi=90°. The fitted orientation functions were then used to accurately calculate the thickness of the thin films. The method proved to be theoretically consistent. The residual strain measurements completed on the thin films were determined using the sin²x method of analysis. Strains were determined from x-ray peak shifts and then plotted against sin²x. Tensile stresses were found to be present in the titanium sample while compressive stresses were present in the molybdenum sample. Lattice expansions were recorded in both film samples (as received condition). These expansions were correlated to the presence of argon as reported by previous authors. Other effects which could have produced additional residual strains were also noted (i.e., thermally induced dislocations, etc.). / Master of Science

One taphonomic problem plaguing archaeologists and physical anthropologists, whether their research is in North American cultures or hominid sites in Africa, is the difficulty in distinguishing bone altered by burning and heating from bone altered by soil processes. Archaeologists working to understand the recent prehistory of the Southern Oregon Coast face the same challenge. Two relatively new tools were investigated to determine their usefulness to resolving this problem. These are scanning electron microscopy (SEM) and X-ray diffraction (XRD). SEM has been well-tested in African sites and experimental studies to identify hominid-created cut marks on bone and to reconstruct heating temperatures of burnt bone. However, SEM and its ability to sample chemistry, as well as XRD's ability to detect diagentic alteration in bone minerals, have not been tested on material from coastal Oregon. The purpose of this research was: (1) to test these methods to see whether they could distinguish between burning and soil alteration, using cervid bone from site 35CS43 near Bandon, Ore., as a test sample, and (2) to see whether the result, paired with archaeological, ethnographic, taphonomic and faunal evidence, could be used to understand how the Coquille were procuring, processing and cooking cervids as insights into their adaptation. The outcome suggests that SEM and XRD, without use of other evidence, are unable to distinguish between burning and soil alteration because the similarities between the two lie not only in changes to the bone's macrostructure (discoloration) but also in bone chemistry, where it was hoped differences could be found. However, these techniques, when paired with the other lines of evidence, did provide insights in understanding the taphonomy and the Coquille's use of cervids - the interaction of bone and soil; the extent of mimicry between burning and soil alteration; and ultimately that discoloration of cervid bone at 35CS43 was likely due to soil alteration, that burning as the result of fire roasting was most likely not occurring at the site, and that the Coquille employed other methods of cooking. / Graduation date: 2003

Cystic fibrosis (CF) is an inherited disease with symptoms mainly in the respiratory tract. The airway epithelium is covered with a thin layer of fluid, the airway surface liquid (ASL). The volume and composition of ASL are important in the pathogenesis of cystic fibrosis. The composition of ASL was determined. Firstly, pig airways were analyzed by X-ray microanalysis in the frozen-hydrated state. Secondly, small Sephadex beads were left to absorb the ASL in situ and were analyzed by X-ray microanalysis. The Na and Cl concentrations in the ASL of the pig were close to those of these ions in serum. Rat tracheal ASL was hypotonic. However, rat nasal fluid was hypertonic with an extremely high concentration of K. The composition of the ASL could be influenced by pharmacological stimulation. The development of transgenic mouse models for CF may help to develop therapies for the disease. The composition of mouse ASL was investigated using different collection techniques. (1) beads mounted on filter paper, (2) beads randomly spread over the airway epithelium, and (3) beads spread over the epithelium with a syringe. No significant difference could be detected between these techniques, and mouse ASL was hypotonic. Calibration curves had to be made for each element of interest. Nasal fluid from healthy human volunteers was collected with: (1) a pipette, (2) filter paper, (3) cotton wool, or (4) Sephadex beads. Collection on filter paper and equilibration with Sephadex beads gave reliable results. The Na and Cl concentrations in nasal fluid of control subjects were about the same as in serum, but the K concentration was higher. Rhinitis or primary ciliary dyskinesia patients and CF heterozygotes had abnormally high concentrations of Na and Cl in their nasal fluid (probably due to inflammation of the nasal epithelium), and CF homozygotes had even higher concentrations of Na and Cl.

Soil salinity is one of the major abiotic stresses that decreases agricultural crop production through imposition of both ionic and osmotic stresses. The accumulation of Na⁺ and Cl⁻ in the cytosol to toxic levels inhibits metabolism. Unlike Na⁺, less is known about Cl⁻ uptake and transport in plants. Grapevine is moderately sensitive to salinity and accumulation of toxic levels of Cl⁻ in leaves is the major reason for salt-induced symptoms. In this study Cl⁻ uptake and transport mechanism(s) were investigated in two grapevine (Vitis sp.) rootstock hybrids differing in salt tolerance: 1103 Paulsen (salt tolerant) and K 51-40 (salt sensitive). Increased external salinity caused high Cl⁻ accumulation in shoots of the salt sensitive K 51-40 in comparison to Paulsen. Measurement of ¹ ⁵ NO₃⁻ net fluxes under high salinity showed that by increasing external Cl⁻ concentrations K 51-40 roots showed reduced NO₃⁻ accumulation. This was associated with increased accumulation of Cl⁻. In comparison to Paulsen, K 51-40 showed reduced NO₃⁻ / Cl⁻ root selectivity with increased salinity, but Paulsen had lower selectivity over the whole salinity range (0-45 mM). In order to examine if root hydraulic and permeability characterisations accounted for differences between varieties, the root pressure probe was used on excised roots. This showed that the osmotic Lpr was significantly smaller than hydrostatic Lpr, but no obvious difference was observed between the rootstocks. The reflection coefficient (σ) values (0.48-0.59) were the same for both rootstocks, and root anatomical studies showed no obvious difference in apoplastic barriers of the main and lateral roots. Comparing the uptake of Cl⁻ with an apoplastic tracer, PTS (3-hydroxy-5, 8, 10-pyrentrisulphonic acid), showed that there was no correlation between Cl⁻ and PTS transport. These results indicated that by-pass flow of salts to the xylem is the same for both rootstocks (10.01±3.03 % and 12.1±1.21 %) and hence pointed to differences in membrane transport to explain difference in Cl⁻ transport to the shoot. ³ ⁶Cl⁻ fluxes across plasma membrane and tonoplast of K 51-40 and Paulsen roots showed that ³ ⁶Cl⁻ influx in root segments of Paulsen was greater than K 51-40 over the first 10 minutes. Unidirectional influx within 10 min loading time showed increases with increases in the external concentrations in both rootstocks but Paulsen had higher influx rate when compared to K 51-40. This appeared to be due to a greater Vmax. There was no significant difference in Km. It was shown that ³ ⁶Cl⁻ accumulation and transport rate to the shoot of K 51-40 was higher than that of Paulsen. Compartmental analysis of ³ ⁶Cl⁻ efflux from intact roots confirmed that the difference in influx observed between the rootstocks was consistent with the results obtained for excised roots, although the values were not exactly the same. It was also shown that the main root of Paulsen had greater contribution to ³ ⁶Cl⁻ uptake than lateral roots. ³ ⁶Cl⁻ fluxes by lateral roots were not significantly different between the rootstocks. Cl⁻ and Na⁺ distribution patterns in different root cell types were determined using the X-ray microanalysis technique. It was shown that Cl⁻ content in the hypodermis and cortical cells was higher than the other cell types in both rootstocks, but overall Cl⁻ content in the root of Paulsen was higher than K 51-40. The pericycle of the main root of Paulsen accumulated more Cl⁻ than K 51-40. It was concluded that Cl⁻ loading to the xylem was different in the rootstocks and Paulsen tended to prevent the xylem Cl⁻ loading process. Lateral roots also displayed opposite behaviour consistent with flux analysis. Membrane potential difference (PD) of the cortical cells showed a rapid and transient depolarization by adding 30 mM NaCl in both rootstocks that was followed by a gradual hyperpolarization. Depolarizations caused by 30 mM Choline-Cl, Na-MES and NaCl measured by the root surface potential method showed that Choline-Cl in K 51-40 and Na-MES in Paulsen caused greater depolarization than that of Na-MES in K 51-40 and Choline-Cl in Paulsen respectively. Assuming that PD measured in this method was the trans-root potential (TRP), it was concluded that the higher depolarization by Choline-Cl in K 51-40 can be due to higher Cl⁻ efflux rate to the xylem. Two different mechanisms were also detected for Cl⁻ transport: HATS which was observed in the range of 0.5-5 mM and a LATS in the range of 10-30 mM of the external NaCl concentration. This was consistent with the concentration dependence of Cl⁻ influx. In conclusion, evidence obtained from different experiments of this study indicated that in the grapevine rootstocks (Paulsen and K 51-40) Cl⁻ was mostly transported through the symplastic pathway. From ECl values determined for the rootstocks by the Nernst equation, a proton-driven transport system was responsible for Cl⁻ transport in both the HATS and LATS range of external NaCl concentrations. The rate of Cl⁻ transport from the root to shoot (xylem loading) was the major difference in Cl⁻ transport between the rootstocks in terms of salinity tolerance. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339051 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Soil salinity is one of the major abiotic stresses that decreases agricultural crop production through imposition of both ionic and osmotic stresses. The accumulation of Na⁺ and Cl⁻ in the cytosol to toxic levels inhibits metabolism. Unlike Na⁺, less is known about Cl⁻ uptake and transport in plants. Grapevine is moderately sensitive to salinity and accumulation of toxic levels of Cl⁻ in leaves is the major reason for salt-induced symptoms. In this study Cl⁻ uptake and transport mechanism(s) were investigated in two grapevine (Vitis sp.) rootstock hybrids differing in salt tolerance: 1103 Paulsen (salt tolerant) and K 51-40 (salt sensitive). Increased external salinity caused high Cl⁻ accumulation in shoots of the salt sensitive K 51-40 in comparison to Paulsen. Measurement of ¹ ⁵ NO₃⁻ net fluxes under high salinity showed that by increasing external Cl⁻ concentrations K 51-40 roots showed reduced NO₃⁻ accumulation. This was associated with increased accumulation of Cl⁻. In comparison to Paulsen, K 51-40 showed reduced NO₃⁻ / Cl⁻ root selectivity with increased salinity, but Paulsen had lower selectivity over the whole salinity range (0-45 mM). In order to examine if root hydraulic and permeability characterisations accounted for differences between varieties, the root pressure probe was used on excised roots. This showed that the osmotic Lpr was significantly smaller than hydrostatic Lpr, but no obvious difference was observed between the rootstocks. The reflection coefficient (σ) values (0.48-0.59) were the same for both rootstocks, and root anatomical studies showed no obvious difference in apoplastic barriers of the main and lateral roots. Comparing the uptake of Cl⁻ with an apoplastic tracer, PTS (3-hydroxy-5, 8, 10-pyrentrisulphonic acid), showed that there was no correlation between Cl⁻ and PTS transport. These results indicated that by-pass flow of salts to the xylem is the same for both rootstocks (10.01±3.03 % and 12.1±1.21 %) and hence pointed to differences in membrane transport to explain difference in Cl⁻ transport to the shoot. ³ ⁶Cl⁻ fluxes across plasma membrane and tonoplast of K 51-40 and Paulsen roots showed that ³ ⁶Cl⁻ influx in root segments of Paulsen was greater than K 51-40 over the first 10 minutes. Unidirectional influx within 10 min loading time showed increases with increases in the external concentrations in both rootstocks but Paulsen had higher influx rate when compared to K 51-40. This appeared to be due to a greater Vmax. There was no significant difference in Km. It was shown that ³ ⁶Cl⁻ accumulation and transport rate to the shoot of K 51-40 was higher than that of Paulsen. Compartmental analysis of ³ ⁶Cl⁻ efflux from intact roots confirmed that the difference in influx observed between the rootstocks was consistent with the results obtained for excised roots, although the values were not exactly the same. It was also shown that the main root of Paulsen had greater contribution to ³ ⁶Cl⁻ uptake than lateral roots. ³ ⁶Cl⁻ fluxes by lateral roots were not significantly different between the rootstocks. Cl⁻ and Na⁺ distribution patterns in different root cell types were determined using the X-ray microanalysis technique. It was shown that Cl⁻ content in the hypodermis and cortical cells was higher than the other cell types in both rootstocks, but overall Cl⁻ content in the root of Paulsen was higher than K 51-40. The pericycle of the main root of Paulsen accumulated more Cl⁻ than K 51-40. It was concluded that Cl⁻ loading to the xylem was different in the rootstocks and Paulsen tended to prevent the xylem Cl⁻ loading process. Lateral roots also displayed opposite behaviour consistent with flux analysis. Membrane potential difference (PD) of the cortical cells showed a rapid and transient depolarization by adding 30 mM NaCl in both rootstocks that was followed by a gradual hyperpolarization. Depolarizations caused by 30 mM Choline-Cl, Na-MES and NaCl measured by the root surface potential method showed that Choline-Cl in K 51-40 and Na-MES in Paulsen caused greater depolarization than that of Na-MES in K 51-40 and Choline-Cl in Paulsen respectively. Assuming that PD measured in this method was the trans-root potential (TRP), it was concluded that the higher depolarization by Choline-Cl in K 51-40 can be due to higher Cl⁻ efflux rate to the xylem. Two different mechanisms were also detected for Cl⁻ transport: HATS which was observed in the range of 0.5-5 mM and a LATS in the range of 10-30 mM of the external NaCl concentration. This was consistent with the concentration dependence of Cl⁻ influx. In conclusion, evidence obtained from different experiments of this study indicated that in the grapevine rootstocks (Paulsen and K 51-40) Cl⁻ was mostly transported through the symplastic pathway. From ECl values determined for the rootstocks by the Nernst equation, a proton-driven transport system was responsible for Cl⁻ transport in both the HATS and LATS range of external NaCl concentrations. The rate of Cl⁻ transport from the root to shoot (xylem loading) was the major difference in Cl⁻ transport between the rootstocks in terms of salinity tolerance. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339051 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

<p>Cystic fibrosis (CF) is the most common lethal genetic disease in the white population. It is due to mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a protein that functions mainly as a cAMP-activated chloride channel. The disease impairs ion and water transport in epithelia-lined organs such as airways, digestive tract, reproductive epithelium and sweat glands. At present the only therapy is symptomatic and development of curative treatment depends on uncovering the links between the defective CFTR and the disease, as well as on improving end-point measurements. </p><p>A method has been established for studying ion transport in an easily accessible cell type (nasal epithelial cells) from normal and cystic fibrosis patients by X-ray microanalysis. This method represents a rather simple and direct way of measuring simultaneously several chemical elements of biological interest.</p><p>Studies of chloride transport by means of a fluorescent indicator (MQAE) in nasal epithelial cells from CF patients showed that the phenotype cannot exclusively be explained by the CFTR activity in patients with severe genotype. </p><p>A common Portuguese CFTR mutation (A561E) causes protein mislocalization in the endoplasmic reticulum similar to the most common CF mutation (ΔF508) and thus it should be possible to treat it with the same pharmacological strategies.</p><p>Chronic treatment of CF airway epithelial cells with nanomolar concentrations of colchicine increased the chloride efflux via chloride channels other than CFTR, strengthening the notion that colchicine could be beneficial to CF patients.</p><p>Successful <i>in vitro </i>transfection of CF airway epithelial cells with cationic vectors was possible with short incubation times. Heparin added at the end of the transfection incubation time could help to maintain the viability of the cells, without interfering with the transfection efficiency. It seems possible that heparin could be an adjuvant for non-viral mediated gene therapy.</p>

Cystic fibrosis (CF) is the most common lethal genetic disease in the white population. It is due to mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a protein that functions mainly as a cAMP-activated chloride channel. The disease impairs ion and water transport in epithelia-lined organs such as airways, digestive tract, reproductive epithelium and sweat glands. At present the only therapy is symptomatic and development of curative treatment depends on uncovering the links between the defective CFTR and the disease, as well as on improving end-point measurements. A method has been established for studying ion transport in an easily accessible cell type (nasal epithelial cells) from normal and cystic fibrosis patients by X-ray microanalysis. This method represents a rather simple and direct way of measuring simultaneously several chemical elements of biological interest. Studies of chloride transport by means of a fluorescent indicator (MQAE) in nasal epithelial cells from CF patients showed that the phenotype cannot exclusively be explained by the CFTR activity in patients with severe genotype. A common Portuguese CFTR mutation (A561E) causes protein mislocalization in the endoplasmic reticulum similar to the most common CF mutation (ΔF508) and thus it should be possible to treat it with the same pharmacological strategies. Chronic treatment of CF airway epithelial cells with nanomolar concentrations of colchicine increased the chloride efflux via chloride channels other than CFTR, strengthening the notion that colchicine could be beneficial to CF patients. Successful in vitro transfection of CF airway epithelial cells with cationic vectors was possible with short incubation times. Heparin added at the end of the transfection incubation time could help to maintain the viability of the cells, without interfering with the transfection efficiency. It seems possible that heparin could be an adjuvant for non-viral mediated gene therapy.

Two problems hampering efforts to produce salt-tolerant plants through constitutive expression of transgenes include: 1. Spatial control. Particular cell-types must respond specifically to salt stress to minimise the amount of Na⁺ delivered to the shoot; and, 2. Temporal control. Transgenes are typically expressed in plants at similar levels through time, irrespective of the stress encountered by the plant, which may exacerbate pleiotropic effects and means that, particularly in low-stress conditions, costly and/or detrimental metabolic processes may be active, thus reducing yield. To address these issues, Gateway® destination vector constructs were developed combining the GAL4 UAS (upstream activating sequence) with the ethanol-inducible gene expression system to drive inducible cell-specific expression of Na⁺ transporter transgenes (or to silence salt transporter transgenes inducibly and cell-specifically). Rice (Oryza sativa L. cv. Nipponbare) GAL4-GFP enhancer trap lines (Johnson et al., 2005: Plant J. 41, 779-789) that express GAL4 and GFP specifically in either the root epidermis or xylem parenchyma (and therefore ‘trap’ cell-type specific enhancer elements) were transformed with this GAL4 UAS – ethanol switch construct, thereby allowing both spatial and temporal control of transgenes. In preliminary experiments, the expression system successfully limited the expression of RFP to specific cell-types after induction with ethanol. Other genes expressed using this system include PpENA1, a Na⁺-extruding ATPase from the moss, Physcomitrella patens, and AtHKT1;1, a Na ⁺ transporter from Arabidopsis thaliana. The two enhancer trap rice lines were also transformed with the GAL4 UAS driving stable expression of AtHKT1;1 and PpENA1 specifically in root epidermal or xylem parenchyma cells. Expression of AtHKT1;1 in root epidermal cells reduced Na⁺ accumulation in the shoots, while expression in the root xylem parenchyma appeared to have little effect on shoot Na⁺ accumulation. Using cryo-scanning electron microscopy (SEM) X-ray microanalysis, the outer cells of the roots of the line expressing AtHKT1;1 in the epidermal cells were found to accumulate higher levels of Na⁺ than the parental enhancer trap line. Additionally, this line had decreased unidirectional ²²Na⁺ influx. Similar results were observed for plants expressing AtHKT1;1 driven by the CaMV 35S / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1325289 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008