The high affinity sodium/glucose cotransporter (SGLT1) couples transport of Na+ and glucose. Investigation of the structure/function relationships of the sodium/glucose transporter (SGLT1) is crucial to understanding co-transporter mechanism. In the first project, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulphonate (MTS) derivatives to test whether predicted TM IV participates in sugar binding. Charged and polar residues and glucose/galactose malabsorption (GGM) missense mutations in TM IV were replaced with cysteine. Mutants exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. The results from mutants T156C and K157C suggest that TM IV participates in sugar interaction with SGLT1. This work has been published in Am J Physiol Cell Physiol 295 (1), C64-72, 2008. The crystal structure of Vibrio parahaemolyticus SGLT (vSGLT) was recently published (1) and showed discrepancy with the predicted topology of mammalian SGLT1 in the region surrounding transmembrane segments IV-V. Therefore, in the second project, we investigated the topology in this region, thirty-eight residues from I143 to A180 in the N-terminal half of rabbit SGLT1 were individually replaced with cysteine and then expressed in COS-7 cells or Xenopus laevis oocytes. Based on the results from biotinylation of mutants in intact COS-7 cells, MTSES accessibility of cysteine mutants expressed in COS-7 cells, effect of substrate on the accessibility of mutant T156C in TM IV expressed in COS-7 cells, and characterization of cysteine mutants in TM V expressed in Xenopus laevis oocytes, we suggest that the region including residues 143-180 forms part of the Na+- and sugar substrate-binding cavity. Our results also suggest that TM IV of mammalian SGLT1 extends from residue 143-171 and support the crystal structure of vSGLT. This work has been published in Biochem Biophys Res Commun 378 (1), 133-138, 2009 Previous studies established that mutant Q457C human SGLT1 retains full activity, and sugar translocation is abolished in mutant Q457R or in mutant Q457C following reaction with methanethiosulfonate derivatives, but Na+ and sugar binding remain intact. Therefore, in the third project, we explored the mechanism by which modulation of Q457 abolishes transport, Q457C and Q457R of rabbit SGLT1 expressed in Xenopus laevis oocytes were studied using chemical modification, the two-electrode voltage-clamp technique and computer model simulations. Our results suggest that glutamine 457, in addition to being involved in sugar binding, is a residue that is sensitive to conformational changes of the carrier. This work has been published in Biophysical Journal 96 (2), 748-760, 2009. Taken together our study along with previous biochemical characterization of SGLT1 and crystal structure of vSGLT, we propose a limited structural model that attempts to bring together the functions of substrate binding (Na+ and sugar), coupling, and translocation. We propose that both Na+ and sugar enter a hydrophilic cavity formed by multiple transmembrane helices from both N-terminal half of SGLT1 and C-terminal half of SGLT1, analogous to all of the known crystal structures of ion-coupled transporters (the Na+/leucine transporter, Na+/aspartate transporter and lactose permease). The functionally important residues in SGLT1 (T156 and K157 in TM 4, D454 and Q457 in TM 11) are close to sugar binding sites.

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The growth of an organism involves the proliferation and migration of cells within an extracellular matrix. As a cell surface receptor, the Dag1 gene product dystroglycan links the intracellular cytoskeleton to the extracellular basement membrane in many cells. Thought to act as a structural protein dystroglycan may also participate in signal transduction. This study aims to better understand the role of dystroglycan during kidney morphogenesis. I hypothesised that a lack of dystroglycan in the precursor cells of the kidney could lead to altered kidney growth. Chimaeric mice deficient in dystroglycan were generated to test this hypothesis. A total of 38 chimaeras had genetic contribution and histological analysis performed on their kidneys. Of the chimaeras analysed, only four demonstrated altered kidney morphology. Further histological, immunohistochemical and biochemical studies established whether a link existed between this morphology and a deficiency in dystroglycan. Normal laminar architecture and nephrotic structures of the kidneys suggest that normal kidney organogenesis occurred in the absence of dystroglycan. The pattern and expression level of basement membrane components suggests that normal basement membrane formation also occured in the absence of dystroglycan. Biochemical analysis revealed that although dystroglycan protein levels correlate with the genetic contribution of the chimaeric kidney, it does not correlate with the altered morphology. Ureter blockage causing hydronephrosis can explain the morphology observed. A deficiency of dystroglycan in the ureter may in turn have caused this blockage. These findings suggest that dystroglycan is not necessary for kidney organogenesis, since kidney development occurred normally in all 38 chimaeric animals irrespective of genetic contribution.

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Membrane biofilm reactors (MBfR) utilize membrane fibers for bubble-less transfer of gas by diffusion and provide a surface for biofilm development. Nitrogen removal was attempted using MBfR in various configurations - nitrification, denitrification and consecutive nitrification and denitrification. Effects of loading rate and dissolved oxygen on nitrification performance were primarily investigated in a stand-alone nitrifying MBfR. Specific nitrification rate increased linearly with specific loading rate, up to the load of 3.5 g N/m²d. Beyond that load, substrate diffusion limitation inhibited further increase of specific nitrification rate. 100% oxygen utilization was achievable under limited oxygen supply condition. Effects of mineral precipitation, dissolved oxygen and temperature on hydrogenotrophic denitrification were investigated in a stand-alone denitrifying MBfR. Mineral precipitation, caused by intended pH control, caused the deterioration of denitrification performance by inhibiting the diffusion of hydrogen and nitrate. Operating reactor in various dissolved oxygen conditions showed that the denitrification performance was not affected by dissolved oxygen in MBfR. Optimum temperature of the hydrogenotrophic denitrification system was around 28°C. Total nitrogen removal in a two-step MBfR system incorporating sequential nitrification and hydrogen-driven autotrophic denitrification was investigated in order to achieve nitrogen removal by autotrophic bacteria alone. Long-term stable operation, which proved difficult in previous studies due to excessive biofilm accumulation in autotrophic denitrification systems, was attempted by biofilm control. Nitrification performance was very stable throughout the experimental periods over 200 days. Performance of autotrophic denitrification was maintained stably throughout the experimental periods, however biofilm control by nitrogen sparging was required for process stability. Biofilm thickness was also stably maintained at an average of 270 µm by the gas sparging biofilm control. According to the cost analysis of denitrifying MBfR, hydrogenotrophic denitrification can be an economical tertiary treatment option compared to conventional denitrifying filter although its economic feasibility highly depends on the cost of hydrogen gas. Although this study was conducted in a lab-scale, the findings from this study can be a valuable stepping stone for larger scale application and open the door for system modifications in future.

The purpose of this thesis was to compare ethinylestradiol (EE2) and nitrogen removal in a conventional membrane bioreactor (C-MBR) and a simultaneous nitrification-denitrification membrane bioreactor (SND-MBR). Two MBRs were operated in parallel for 450 days; various MBR operating parameters, total nitrogen removal, and estrogenic activity removal (EA) were measured. The SND-MBR was able to remove 59% of influent TN with an additional 21% removed via sludge wasting; the C-MBR had a TN removal efficiency of only 31%. The C-MBR and SND-MBR removed 57% and 58% of influent EA, respectively. Biodegradation was the dominant removal mechanism for both reactors with KBIO coefficients of 1.5 ± 0.6 and 1.6 ± 0.4 days-1 for the C-MBR and the SND-MBR, respectively. Adsorption removed approximately 1% of influent EA in each reactor. This indicates that SND was able remove greater amounts of TN with no observable impact on EA reduction and membrane operations.

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No description available.

No description available.