To examine the effect of buttermilk fractions on growth, iron transport, and uptake, Caco-2 cells (human colon adenocarcinoma) were grown in a bicameral chamber. The Caco-2 cell culture system is a useful model to study micronutrient utilization in the human enterocyte, because Caco-2 cells continuously differentiate and form a monolayer, which has high polarity, a well-developed brush border, and a tight junction. Iron bioavailabilty in various milks is very different depending upon milk composition. The fat fraction especially is known to be associated with iron absorption, because the fat fraction has milk fat globule membrane (MFGM), which contains bioactive molecules such as sphingolipids. Composition of buttermilk that was concentrated by 10 K molecular sieving (MS) or by bacterial fermentation (Lactococcus latis PN-l) was reduced in lactose concentration and increased in protein concentration. Percent fat in MS buttermilk was concentrated to two times higher than in the original buttermilk (P < 0.05). Growth of Caco-2 cells with molecular sieved (MS) or fermented (FM) buttermilk in the growth medium was not significantly different. Transport and uptake of 59Fe was performed with/without cold iron (1 mmol/L) by iron-depleted or iron-repleted cells. Molecular sieved or fermented buttermilk and ganglioside or sphingomyelin standards with dimethyl sulfoxide (DMSO) were added to the Hank's balance salt solution (HBSS) in the apical chamber. With cold iron, addition of MS and FM buttermilk (1, 2, or 3 percent) increased 59Fe transport across iron-repleted cells (P < 0.01). Without cold iron, ganglioside depressed 59Fe transport (P < 0.01). Uptake of 59Fe was not significantly affected by buttermilk concentrates; however, more effective uptake was shown across iron-depleted cells. It is not clear from these studies that buttermilk fractions or their components influence iron uptake or transport by Caco-2 cell cultures.

Seagrass communities are composed of numerous organisms that depend on water-column nutrients for metabolic processes. The rate at which these organisms remove a nutrient from the water column can be controlled by physical factors such as hydrodynamic regime or by biological factors such as speed of enzyme reactions. The impact of hydrodynamic regime on rates of nutrient uptake for seagrass (Thalassia testudinum) communities and for organisms that comprise the community (seagrass, epiphytes, phytoplankton, and microphytobenthos) was quantified in a series of field flume experiments employing the use of 15N-labeled ammonium and nitrate. Rates of ammonium uptake for the entire community and for seagrass leaves and epiphytes were significantly dependent on bulk velocity, bottom shear stress, and the rate of turbulent energy dissipation. Relationships between uptake rates and these parameters were consistent with mass-transfer theory and suggest that the effect of water flow on ammonium uptake is the same for the benthos as a whole and for the organisms that form the canopy. In addition, epiphytes on the surface of T. testudinum leaves were shown to depress leaf uptake by an amount proportional to the area of the leaf covered by epiphytes. Water flow influenced rates of nitrate uptake for the community and the epiphytes; however, uptake rates were depressed relative to those for ammonium suggesting that uptake of nitrate was also affected by biological factors such as enzyme activity. Epiphytes reduced uptake of nitrate by the leaves; however, the amount of reduction was not proportional to the extent of epiphyte cover, which provided further evidence that nitrate uptake by T. testudinum leaves was biologically limited. As an additional component of the research, hydrodynamic regime of a mixed seagrass and coral community in Florida Bay was characterized using an acoustic Doppler velocimeter. Hydrodynamic parameters estimated from velocity data were used in mass-transfer equations to predict nutrient uptake by the benthos over a range of water velocity. Measured rates of uptake from field flume experiments conducted in the same community confirmed that hydrodynamic data could be used to accurately predict nutrient transport to the benthos under natural flow conditions.

Siderophores are low molecular weight, iron chelating compounds produced by many bacteria for uptake of iron in case of iron scarcity. Vicibactin is a trihydroxamate type siderophore produced by Rhizobium leguminosarum bv. trifolii ATCC 14479. This work focuses on identifying an outer membrane receptor involved in the transport of vicibactin. We have confirmed the presence of the putative fhuA gene in R. leguminosarum bv. trifolii ATCC 14479. This bacteria shows mutualistic symbiosis with the red clover plant Trifoliium prantense. Leghemoglobin, with its cofactor heme is present in the plant root nodules that surrounds the infecting organism present in the nodules. This work attempts to elucidate the ability of Rhizobium leguminosarum bv. trifolii ATCC 14479 to utilize heme-bound iron and genes involved in the transport. We have also elucidated the role of energy transducing proteins TonB- ExbB-ExbD on the heme-bound iron uptake system.

Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X7 receptor. These responses include opening of a cation selective channel/pore which allows entry of the fluorescent dye, ethidium+ and activation of a membrane metalloprotease which sheds the adhesion molecule L-selectin. In this thesis, the surface expression of P2X7 receptors was measured in normal leucocytes, platelets and B-CLL lymphocytes and compared with their functional responses. Monocytes showed 4-5 fold greater expression of P2X7 than B-, T- and NK- lymphocytes, while P2X7 expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed surface P2X7 at about the same level as for B-lymphocytes from normal subjects. P2X7 function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes. However, the ATP-induced uptake of ethidium into the malignant B-lymphocytes in 3 patients was low or absent. The lack of P2X7 function in these B-lymphocytes was confirmed by the failure of ATP to induce Ba2+ uptake into their lymphocytes. This lack of function of the P2X7 receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule which directs the recirculation of lymphocytes from blood into the lymph node. To study a possible genetic basis of non-functional P2X7 receptor, we sequenced DNA coding for the carboxyl terminal tail of P2X7. In 33 of 130 normal subjects a heterozygous nucleotide substitution (1513A--C) was found while 3 subject carried the homozygous substitution which codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this 496Glu--Ala polymorphism demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the 496Glu--Ala homozygote subject expressed non-functional receptor while heterozygotes showed P2X7 function which was half that of wild type P2X7. Results of transfection experiments showed the mutant P2X7 receptor was non-functional when expressed at low receptor density but regained function at a high receptor density. This density-dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma which upregulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with wild type. The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor. Apart from the 496Glu--Ala polymorphism, three other single nucleotide polymorphisms, 155His--Tyr, 348Ala--Thr and 568Ile--Asn were also found in the P2X7 receptor. The site directed mutant cDNA were generated for all 3 polymorphisms and transfected into HEK293 cells to study the impact of these polymorphisms on P2X7 function. Results suggested that Ile568 is important for P2X7 protein trafficking to cell surface. Further study of these two loss-of-function polymorphisms (496Glu--Ala and 568Ile--Asn) may help better understanding of the functional domains in the P2X7 receptor and its role in CLL, lymphoma and infectious diseases. Conclusions: 1.P2X7 receptor is expressed in human leukocytes, including lymphocytes, natural killer cells as well as monocytes, on both surface and intracellular locations. 2.Both the expression and function of P2X7 are highly variable between in human individuals. Non-functional P2X7 receptors are found in some subjects, including both normal subjects and CLL patients, and are often associated with defects in ATP-induced cytotoxicity and L-selectin shedding. 3.Two single nucleotide polymorphisms (SNPs), 496Glu--Ala and 568Ile--Asn, are found at low frequency in the human population and lead to the loss-of-function of P2X7. Both permeabllity function and the downstream effects mediated by P2X7 are affected by these two SNPs. The mechanisms for the loss-of-function differs between the two polymorphisms.

This thesis considers the implications of changes in the supply of resources for photosynthesis, with regard for modes of carbon acquisition employed by aquatic plants of the River Murray. Carbon supplies are inherently more variable for aquatic plants than for those in terrestrial environments, and variations are intensified for plants in semi-arid regions, where water may be limiting. In changeable environments the most successful species are likely to be those with flexible carbon-uptake mechanisms, able to accommodate variations in the supply of resources. Studies were made of plants associated with wetland habitats of the Murray, including Crassula helmsii, Potamogeton tricarinatus, P. crispus and Vallisneria americana. The aim was to elucidate the mechanisms of carbon uptake and assimilation employed, and to determine how flexibility in carbon uptake and/or assimilation physiology affect survival and distribution. Stable carbon isotopes were used to explore the dynamics of carbon uptake and assimilation, and fluorescence was used to identify pathways and photosynthetic capacity. The studies suggest that physiological flexibility is adaptive survival in changeable environments, but probably does not enhance the spread or dominance of these species. V. americana is a known bicarbonate-user, and it is shown here that it uses the Crassulacean Acid Metabolism (CAM) photosynthetic pathway under specific conditions (high light intensity near the leaf tips) concurrently with HCO[subscript]3 - uptake, while leaves deeper in the water continue to use the C[subscript]3 pathway, with CO₂ as the main carbon source. However, V. americana does not use CAM when under stress, such as exposure to high light and temperature. The diversity of carbon uptake and assimilation mechanisms in this species may explain its competitive ability in habitats associated with the Murray. In this way it is able to maximise use of light throughout the water column. In shallow, warm water, where leaves are parallel to the surface, CAM ability is likely to be induced along the length of the leaf, allowing maximal use of carbon and light. The amphibious C. helmsii is shown to use CAM on submergence, even where water levels fluctuate within 24 hours. This allows continued photosynthesis in habitats where level fluctuations prevent access to atmospheric CO₂. It appears that stable conditions are most favourable for growth and dispersal, and that the spread of C. helmsii is mainly by the aerial form. Carbon uptake by P. tricarinatus under field conditions is compared with that of P. crispus to demonstrate differences in productivity associated with aqueous bicarbonate and atmospheric CO₂ use. P. tricarinatus uses HCO[subscript]3 - uptake to promote growth toward the surface, so that CO₂ can be accessed by floating leaves. Atmospheric contact provides access to light and removes the limitation of aqueous diffusive resistance to CO₂, thereby increasing photosynthetic capacity above that provided by submerged leaves. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1320380 / Thesis (Ph.D) -- University of Adelaide, School of Earth and Environmental Sciences, 2008

Four tumour active trinuclear complexes: DH4Cl: [{trans-PtCl(NH3)2}2m-{trans-Pd( NH3)2(H2N(CH2)4NH2)2]Cl4, DH5Cl: [{trans-PtCl(NH3)2}2m-{trans-Pd( NH3)2(H2N(CH2)5NH2)2]Cl4, DH6Cl: [{trans-PtCl(NH3)2}2m-{trans-Pd( NH3)2(H2N(CH2)6NH2)2]Cl4, DH7Cl: [{trans-PtCl(NH3)2}2m-{trans-Pd(NH3)2-( H2N(CH2)7NH2)2]Cl4 and one dinuclear complex DHD: [{trans-PtCl(NH3)2}�-{ H2N(CH2)6NH2}{trans-PdCl(NH3)2]Cl(NO3), have been prepared and characterised based on elemental analyses, IR, Raman, mass and 1 H NMR spectral measurements. For the trinuclear complexes, the synthesis has been carried out using a step-up method branching out from the central palladium unit. A purity of about 95% has been obtained by repeated dissolution and precipitation. The activity against human cancer cell lines including ovary cell lines: A2780, A2780 cisR , A2780 ZD0473R , non small lung cell line: NCI-H640 and melanoma: Me-10538 have been determined based on MMT assay. Cell uptakes, DNA-binding have been determined for ovary cell lines: A2780, A2780 cisR . The nature of interaction with pBR322 plasmid DNA and ssDNA has been studied for trinuclear complexes DH4Cl, DH5Cl, DH6Cl and DH7Cl and the dinuclear complex DHD. Interaction of DH6Cl with adenine and guanine has also been studied by HPLC. The compounds are found to exhibit significant anticancer activity against cancer cell lines especially ovarian cancer cell lines: A2780, A2780 cisR and A2780 ZD0473R . DH6Cl in which the linking diamine has six carbon atoms is found to be the most active compound. As the number of carbon atoms in thelinking diamine is changed from the optimum value of six, the activity is found to decrease, illustrating the structure-activity relationship. The increase in uptake of the trinuclear complexes in A2780 cell line with the increase in size of the linking diamine coupled with the low molar conductivity values found for the solutions of the compounds suggest that the compounds would remain in solution as undissociated �molecules� and hence could cross the cell membrane by passive diffusion. Much lower resistance factors for the all the multinuclear compounds including DHD as applied to A2780 cisR cell line, as compared to that for cisplatin, suggest that the compounds are able to overcome multiple mechanisms of resistance operating in the cell line. All of the multinuclear complexes are expected to form long-range interstrand GG adducts with DNA, causing irreversible global changes in the DNA conformation but unlike cisplatin do not cause sufficient DNA bending to be recognized by HMG 1 protein. Increasing prevention of BamH1 digestion with the increase in concentration of the multinuclear compounds also provide support to the idea that the compounds because of the formation of a plethora of interstrand GG adducts are able to cause irreversible changes in DNA conformation. The results of the study show that indeed new trinuclear tumour active compounds can be found by replacing the central platinum unit in BBR3464 with other suitable metal units.

Oxidative damage has been suggested to play a key role in the pathogenesis of atherosclerosis and other cardiovascular disease. Increased free radical production induced by oxidative stress can oxidise low density lipoproteins, activates platelets, induces endothelial dysfunction and disturbs glucose transport by consuming endogenous antioxidants. Using a combination, of in vitro and in vivo experimental models, the primary aims of the studies undertaken for this thesis were to examine whether different antioxidants could negate risk factors leading to thrombosis, atherosclerosis and other cardiovascular diseases. The studies utilised the mechanisms involved in platelet activity and glucose uptake by skeletal muscle myotubes. The first study determined if olive leaf extract would attenuate platelet activity in healthy human subjects. Blood samples (n=11) were treated with five different concentrations of extract of Olea europaea L. leaves ranging from 5.4£gg/mL to 54£gg/mL. A significant reduction in platelet activity (pless than0.001) and ATP release from platelets (p=0.02) was observed with 54£gg/mL olive leaf extract. The next crossover study compared the effect of exercise and antioxidant supplementation on platelet function between trained and sedentary individuals. An acute bout of 1 hour exercise (sub maximal cycling at 70% of VO2max) was used to induce oxidative stress in 8 trained and 8 sedentary male subjects, before and after one week supplementation with 236 mg/day of cocoa polyphenols. Baseline platelet count and ATP release increased significantly (pless than0.05) after exercise in all subjects. Baseline platelet numbers in the trained were higher than in the sedentary (235¡Ó37 vs. 208¡Ó34 x109/L, p less than 0.05), whereas platelet activation in trained subjects was lower than sedentary individuals (51¡Ó6 vs. 59¡Ó5%, p less than0.05). Seven days of cocoa polyphenol supplementation did not change platelet activity compared to the placebo group. The third study determined the effect of 5 weeks of either 100mg/day £^-Tocopherol (n=14), 200mg/d £^-Tocopherol (n=13) or placebo (n=12) on platelet function, lipid profile and the inflammatory marker C-reactive protein. Blood £^-tocopherol concentrations increased significantly (pless than0.05) relative to dose. Both doses attenuated platelet activation (pless than0.05). LDL cholesterol, platelet aggregation and mean platelet volume were decreased by 100mg/d £^-tocopherol (all pless than0.05). The final study determined the effect of glucose oxidase induced oxidative stress and £^-tocopherol treatment on glucose transport and insulin signalling in cultured rat L6 muscle cells. One hour treatment with 100mU/mL glucose oxidase significantly decreased glucose uptake both with and without 100nM insulin stimulation (pless than0.05). Pre-treatment with 100ƒÝM and 200ƒÝM £^-tocopherol partially protected cells from the effect of glucose oxidase, whereas 200ƒÝM £^-tocopherol restored both basal and insulin stimulated glucose transport to control levels. Glucose oxidase-induced oxidative stress did not impair basal or insulin stimulated phosphorylation of Akt or AS160, but 200ƒÝM £^-tocopherol improved insulin-stimulated phosphorylation of these proteins. In summary, the results from the studies undertaken for this thesis provide evidence that antioxidant supplementation maintains normal platelet function, exerts a positive effect on blood lipid profile and improves glucose uptake in normal healthy asymptomatic population as well as under conditions of induced oxidative stress. Antioxidants including foods rich in cocoa, olive and gamma tocopherol have the potential to combat oxidative stress induced risk factors leading to cardiovascular diseases.

<p>Metal contaminated stormwater, i.e. surface runoff in urban areas, can be treated in percolation systems, ponds, or wetlands to prevent the release of metals into receiving waters. Plants in such systems can, for example, attenuate water flow, bind sediment, and directly accumulate metals. By these actions plants affect metal mobility. This study aimed to examine the accumulation of Zn, Cu, Cd, and Pb in roots and shoots of plant species common in stormwater areas. Furthermore, submersed plants were used to examine the fate of metals: uptake, translocation, and leakage. Factors known to influence metal accumulation, such as metal ion competition, water salinity, and temperature, were also examined. The following plant species were collected in the field: terrestrial plants – <i>Impatiens parviflora</i>, <i>Filipendula ulmaria</i>, and <i>Urtica dioica</i>; emergent plants –<i> Alisma-plantago aquatica</i>, <i>Juncus effusus</i>, <i>Lythrum salicaria</i>, <i>Sagittaria sagittifolia</i>, and <i>Phalaris arundinacea</i>; free-floating plants – <i>Lemna gibba</i> and <i>Lemna minor</i>; and submersed plants – <i>Elodea canadensis</i> and <i>Potamogeton natans</i>. Furthermore, the two submersed plants, <i>E. canadensis</i> and <i>P. natans</i>, were used in climate chamber experiments to study the fate of the metals in the plant–water system.</p><p>Emergent and terrestrial plant species accumulated high concentrations of metals in their roots under natural conditions but much less so in their shoots, and the accumulation increased further with increased external concentration. The submersed and free-floating species accumulated high levels of metals in both their roots and shoots. Metals accumulated in the shoots of <i>E. canadensis</i> and <i>P. natans</i> derived mostly from direct metal uptake from the water column.</p><p>The accumulation of Zn, Cu, Cd, and Pb in submersed species was in general high, the highest concentrations being measured in the roots, followed by the leaves and stems, <i>E. canadensis</i> having higher accumulation capacity than <i>P. natans</i>. In <i>E. canadensis</i> the Cd uptake was passive, and the accumulation in dead plants exceeded the of living with time. The capacity to quickly accumulate Cd in the apoplast decreased with successive treatments. Some of the Cd accumulated was readily available for leakage. In <i>P. natans,</i> the presence of mixtures of metal ions, common in stormwater, did not alter the accumulation of the individual metals compared to when presented separately. It is therefore, proposed that the site of uptake is specific for each metal ion. In addition cell wall-bound fraction increased with increasing external concentration. Further, decreasing the temperature from 20ºC to 5ºC and increasing the salinity from 0‰ to 5‰ S reduced Zn and Cd uptake by a factor of two.</p><p>In <i>P. natans</i> the metals were not translocated within the plant, while in<i> E. canadensis </i>Cd moved between roots and shoots. Thus,<i> E. canadensis</i> as opposed to <i>P. natans</i> may increase the dispersion of metals from sediment via acropetal translocation. The low basipetal translocation implies that neither <i>E. canadensis</i> nor <i>P. natans</i> will directly mediate the immobilisation of metal to the sediment via translocation.</p><p>To conclude, emergent and terrestrial plant species seem to enhance metal stabilization in the soil/sediment. The submersed plants, when present, slightly increase the retention of metals via shoot accumulation.</p>

<p>The use of nano- and micro-particulate carriers as delivery systems for oral vaccines has been under investigation for several decades. Surprisingly little is known of their uptake in the human intestine, despite the fact that substantial improvement is required to achieve adequate immune responses in man after oral administration. </p><p>In this thesis, various aspects of particle transcytosis across the human intestinal epithelium were studied, in order to identify strategies for improved uptake of nano- and micro-particulate drug delivery systems. </p><p>The follicle associated epithelium (FAE) overlying Peyer´s patches contains M-cells, which have an increased capacity for uptake of particulate antigens. Therefore, a model of human FAE was developed to study mechanisms of particle uptake and transport.</p><p>Receptors that could be used for targeting to the FAE had previously not been identified in humans. By use of the model FAE, two new targets were identified on human intestinal FAE; CD9 and β1-integrin. Furthermore, studies of isolated human intestinal tissue showed that an integrin-adherent peptide motif, RGD, could be utilized to achieve selective and improved transport of nanoparticles into human Peyer´s patches.</p><p>Studies of factors influencing intestinal particle uptake and transcytosis revealed that two cytokines, TNF-α and LTα1/β2, but also one growth factor, TGF-β1, induced uptake of particles in Caco-2 cells and transcytosis of particles in the model FAE. Furthermore, it was shown that an enteric bacterium, Yersinia Pseudotuberculosis, could trigger uptake and transcytosis of particles across model absorptive epithelial cells.</p><p>In conclusion, this thesis provides a platform for further investigations of particle transcytosis across the human intestinal epithelium. The identification of two new proteins with increased expression in human FAE and a targeting sequence that improves particle uptake into Peyer’s patches, gives new hope for the development of subunit oral vaccines.</p>

<p>Interaction between mercury and selenium has previously been observed both in man and in animals. The aim of this work was to study expressions of interaction between mercury and selenium in human K-562 cells. Inorganic and organic forms of mercury and selenium were used and cells were either pre-treated with selenium or simultaneously exposed to selenium and mercury. Concentrations of selenium and mercury chosen were indicated by a study of growth inhibition in the individual compounds: a low concentration of selenium and selenomethionine induced slight cell growth inhibition, while a high concentration resulted in a notable growth inhibition. Two mercury concentrations were chosen: one with minimal toxicity and another with high cell toxicity. In addition, uptake and retention patterns of selenomethionine and selenite differed in both selenocompounds.</p><p>All simultaneous treatments with 3.5 μM methylmercury produced a reduction in cellular mercury with increased selenium concentration. This was particularly obvious in selenite treatments. Growth curves from the simultaneous 3.5 μM methylmercury and selenite treatments indicated protection with increased selenite concentrations. In both exposure protocols, the 5 μM methylmercury treatments were toxic to the cells. </p><p>In both study protocols, cells exposed to selenite and mercuric chloride manifested increased cellular mercury uptake with increased selenium concentration. In all selenite and 35 μM mercuric chloride treatments, no inhibition of growth was observed, while the 50 μM mercuric chloride treatments were toxic to the cells. Selenite-dependent protection was achieved in both exposure protocols when considering the cellular uptake of mercury. With few exceptions, selenomethionine produced similar effects as selenite on mercuric chloride uptake and growth inhibition.</p>