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The Ca²⁺-mobilising messenger NAADP has been shown to play a key role in the regulation of mammalian physiology. Recently, the two-pore channels (TPCs) have been proposed as an NAADP-gated Ca²⁺ channel. Chapter 1 introduces the TPCs as the major candidates in governing NAADP-mediated Ca²⁺-release from acidic stores. Chapter 2 explains the methodologies developed and used. Chapter 3 demonstrates the successful immunopurification of HsTPC2 and its incorporation into an artificial lipid bilayer. K⁺ and Ca²⁺ currents were seen in reponse to nM - μM concentrations of NAADP; with the open probability (P₀) fitting a bell-shaped concentration-response curve. Ligand sensitivity was shown to be regulated by luminal [Ca²⁺], whereby a 20-fold increase in [Ca²⁺] _lumen (10 μM to 200 μM) caused a 100-fold reduction in the EC₅₀ from ≈ 500 nM to 5 nM. Furthermore, a reduction in luminal pH from 7.2 to 4.8 reduced the P₀ but 1 μM Ned-19 inhibiting all channel activity. Chapter 4 investigates the in situ properties of HsTPC2 by the purification and patch clamp of intact lysosomes from cells overexpressing HsTPC2. Three methods of purification were compared: differential centrifugation, whole lysosome immunoprecipitation and magnetic chromatography. Techniques involving lysosomal swelling and whole cell homogenisation were also optimised to ensure minimal contamination by non-lysosomal proteins. Whole lysosome patch clamping revealed NAADP-induced, Ca²⁺-specific currents in response to NAADP, but not cADPR, IP₃ or Ned-19. High concentrations of NAADP (mM) and Ned-19 (μM) showed prolonged ≈ 5 minutes) inhibition of channel activity. Chapter 5 explores the protein-protein interactions of the purified HsTPC2 and identifies a heterodimeric interaction between HsTPC1 and HsTPC2 was further dissected by coimmunoprecipitation, colocalisation and FRET studies. Despite clear evidence that both isoforms independently form homodimers, it is likely that heterodimerisation is a dynamic interaction only seen in a subset of the channel population. Chapter 6 discusses the results obtained in the wider context of cell physiology.

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ABSTRACT The Chloride Intracellular Channel (CLIC) proteins are a family of amphitropic proteins that can convert from soluble to integral membrane forms. CLIC1 is a member of this family that functions as a chloride channel in the plasma and nuclear membranes of cells. Although high-resolution structural data exists for the soluble form of monomeric CLIC1, not much is known about the integral membrane forms’ structure. The exact mechanism and signals involved in the conversion of the soluble form to membrane-inserted form are also not clear. Studies were undertaken in the absence and presence of membrane models. Analysis of the structure and stability of CLIC1 in the absence of membrane investigated the effect of possible signals or triggers that may play a crucial role in the conversion of the soluble form to integral membrane form. Exposing CLIC1 to oxidizing conditions results in the formation of a dimeric form. The CLIC1 dimer was found to be less stable than the monomeric form based on unfolding kinetic studies. The stability of the dimer was also less influenced by salt concentration, compared with the monomer. The effect of pH on the structure of CLIC1 is of physiological relevance since the movement of soluble CLIC1 in the cytoplasm or nucleoplasm toward the membrane will involve the protein being exposed to a lower pH micro-environment. Hydrogen exchange mass spectrometry was used to study the structural dynamics of CLIC1 at pH 7.0 and pH 5.5. At neutral pH, domain II is more stable than the more flexible thioredoxin domain I. The thioredoxin-fold therefore is more likely to unfold and rearrange to insert into membranes. Because of the high stability of domain II this region is probably where the folding nucleus of the protein is. At pH 5.5 it was found that the a1, a3 and a6 helices, which are spatially adjacent to one another across the domain interface, were destabilized. This destabilization may be the trigger for CLIC1 to unfold and rearrange into a membrane insertion-competent form. The role of the primary sequence and unique three-dimensional structure of CLIC1 in membrane insertion was investigated in a bioinformatics-based study that looked at conserved residue features such as hydropathy and charge. Hidden helical propensities and Ncapping motifs in the a1-b2 region were found, which may have important implications for locating putative transmembrane regions. Analysis of the structure and thermodynamics of CLIC1 interacting with membranes investigated changes in secondary structure, tertiary structure, hydrodynamic volume and thermodynamics when CLIC1 is exposed to membrane-mimicking models. The effect of a variety of conditions such as pH and redox, cysteine-modifiying agents (NEM), ligands (GSH), and inhibitors (IAA) on CLIC1 membrane interaction were studied. It was found that CLIC1 interacted with membranes more favourably at lower pH and that NEM completely inhibited CLIC1 interaction with micelles.

The stable isotopic composition of groundwater within a watershed in eastern Iowa was studied in order to understand how water moves through the system. Samples were gathered using multiple observation wells and pore water samplers and then analyzed to determine the δ18O and δ2H of each sample. Shallow pore water is much more variable in its isotopic composition than deeper water and seems to be more greatly affected by evapotranspiration, whereas groundwater below the water table appears to show a stable isotopic signature suggesting the integration of multiple rain events. Other samples of similar depths across the slope of a hill were also used to observe differences across the area. By observing changes over time in the signatures of these samples, it can be seen that the crest of the hill is most greatly influenced by infiltration from precipitation while the side of the hill is influenced more by throughflow. By combining stable isotope analyses, knowledge of the medium through which the water is moving and the general mechanics of a watershed, a more advanced understanding of how water interacts with and moves through the ground can be gained.

To gain an understanding of the transfer of nutrients and trace elements from sediment pore waters to surface waters of eutrophic Lake Rotorua and the early diagenetic processes controlling the transfer, pore water chemistry in the sediments of Lake Rotorua was investigated over a one year period in 2006 by collection of sediment cores on three occasions and deployment of pore water equilibrators on two occasions. Pore water concentrations of Fe2+, Mn2+, S, PO4, NH4, As, Cd, and Pb were analysed. Phosphate and ammonium fluxes to the water column from the sediments were calculated from measured concentration gradients by Fick's law of diffusion. Gas present in the sediments was analysed for composition, and source, and its ebullition rate measured. Anaerobic oxidation of organic matter is indicated by negative Eh values. Sulfate reduction was indicated near the sediment-water interface and releases of Fe2+, Mn2+, PO4 and NH4 into the pore water from particulate material were associated with the reducing conditions. Peaks in concentration of nutrients and elements occurred at the sediment surface over summer and deeper in the pore water profile over the cooler months of May and September. Sampling with peepers at fine scales immediately above the sediment-water interface indicated the presence of a nepheloid layer where elements are actively being recycled. Sulfate reduction appears to occur in the layer above the sediment-water interface, indicating that dissolved oxygen has already been reduced. Phosphorus is possibly being removed by iron and manganese oxide/hydroxide precipitation 5 to 15 cm above the sediment-water interface. Pore water saturation calculations indicate that sulfides may be controlling concentrations of iron and possibly other metals in the pore water by formation of pyrite in the zone of sulfate reduction. Below the zone of sulfate reduction, siderite and vivianite may be precipitating and acting as an additional sink for iron and phosphorus. ii Nutrient release rates based on Fick's law of diffusion indicated 430 tonnes of dissolved phosphorus and 1150 tonnes of ammonium were released to Lake Rotorua's water column in 2006, suggesting nutrient release from the sediments is the dominant flux of nutrients to the water column of Lake Rotorua. Methanogenesis, from acetate fermentation, occurs below the zone of sulfate reduction, where it becomes the dominant process in organic matter degradation. Ebullition of gas was measured at 126 ml m-2 d-1 and this gas was comprised dominantly of methane. Possible remediation techniques that could reduce the internal load of nutrients released from the lake sediments include sediment removal by dredging or capping the sediments with an adsorbent or sealing layer. Capping the sediments could be compromised by ebullition of gas that would disrupt the capped layer, opening up pathways that allow more readily for exchange between pore water nutrients and the water column. Dredging is likely to stimulate the ebullition of most of the trapped gas and result in a rapid efflux of much of the nutrient rich pore water into the lake, however dredging the top 10 to 20 cm of the sediments may partially reduce phosphorus in the pore waters but would not substantially reduce ammonium and fluxes would remain similar to current levels. Improving redox conditions in the sediments could reduce pyrite formation improving phosphorus binding with iron.

Pore pressure exists ubiquitously in the Earth’s subsurface and very often exhibits a cyclic loading on pre-existing faults due to seasonal and tidal changes, as well as the impoundment and discharge of surface reservoirs. The effect of oscillating pore pressure on induced seismicity is not fully understood. This effect exhibits a dynamic variation in effective stresses in space and time. The redistribution of pore pressure as a result of fluid flow and pressure oscillations can cause spatial and temporal changes in the shear strength of fault zones, which may result in delayed and protracted slips on pre-existing fractures. This research uses an experimental approach to investigate the effects of oscillating pore pressure on induced seismicity. With the aid of geophysical techniques, the spatial and temporal distribution of seismic events was reconstructed and analysed. Triaxial experiments were conducted on two types of sandstone, one with low permeability (Fontainebleau sandstone) and the other with high permeability (Darley Dale sandstone). Cyclic pore pressures were applied to the naturally-fractured samples to activate and reactivate the existing faults. The results indicate that the mechanical properties of the sample and the heterogeneity of the fault zone can influence the seismic response. Initial seismicity was induced by applying pore pressures that exceeded the previous maximum attained during the experiment. The reactivation of faults and foreshock sequences was found in the Fontainebleau sandstone experiment, a finding which indicates that oscillating pore pressure can induce seismicity for a longer period of time than a single-step increase in pore pressure. The corresponding strain change due to cyclic pore pressure changes suggests that progressive shearing occurred during the pore pressure cycles. This shearing progressively damaged the existing fault through the wearing of asperities, which in turn reduced the friction coefficient and, hence, reduced the shear strength of the fault. This ‘slow’ seismic mechanism contributed to the prolonged period of seismicity. This study also applied a material forecast model for the estimation of time-to-failure or peak seismicity in reservoir-induced seismicity, which may provide some general guidelines for short-term field case estimations.

Pore pressure exists ubiquitously in the Earth’s subsurface and very often exhibits a cyclic loading on pre-existing faults due to seasonal and tidal changes, as well as the impoundment and discharge of surface reservoirs. The effect of oscillating pore pressure on induced seismicity is not fully understood. This effect exhibits a dynamic variation in effective stresses in space and time. The redistribution of pore pressure as a result of fluid flow and pressure oscillations can cause spatial and temporal changes in the shear strength of fault zones, which may result in delayed and protracted slips on pre-existing fractures. This research uses an experimental approach to investigate the effects of oscillating pore pressure on induced seismicity. With the aid of geophysical techniques, the spatial and temporal distribution of seismic events was reconstructed and analysed. Triaxial experiments were conducted on two types of sandstone, one with low permeability (Fontainebleau sandstone) and the other with high permeability (Darley Dale sandstone). Cyclic pore pressures were applied to the naturally-fractured samples to activate and reactivate the existing faults. The results indicate that the mechanical properties of the sample and the heterogeneity of the fault zone can influence the seismic response. Initial seismicity was induced by applying pore pressures that exceeded the previous maximum attained during the experiment. The reactivation of faults and foreshock sequences was found in the Fontainebleau sandstone experiment, a finding which indicates that oscillating pore pressure can induce seismicity for a longer period of time than a single-step increase in pore pressure. The corresponding strain change due to cyclic pore pressure changes suggests that progressive shearing occurred during the pore pressure cycles. This shearing progressively damaged the existing fault through the wearing of asperities, which in turn reduced the friction coefficient and, hence, reduced the shear strength of the fault. This ‘slow’ seismic mechanism contributed to the prolonged period of seismicity. This study also applied a material forecast model for the estimation of time-to-failure or peak seismicity in reservoir-induced seismicity, which may provide some general guidelines for short-term field case estimations.

Streptococcus agalactiae CAMP factor is a protein exotoxin that contains 226 amino acid residues and forms oligomeric pores on susceptible cell membranes and liposomes. In this study, fragments of CAMP factor were created and recombinantly expressed to identify functional domains that are involved in membrane binding, oligomerization, and membrane insertion. Altogether, six truncated forms of CAMP factor were created and assayed. CAMP1-113, CAMP1-170, CAMP57-226, and CAMP171-226 showed different levels of hemolytic activity. CAMP1-56 and CAMP114-226 did not show hemolytic activity or oligomerization ability, but showed binding ability. CAMP114-226 inhibited the hemolytic activity of wild-type CAMP factor, most likely through ‘one-sided’ oligomerization. From the comparison of these fragments, it emerges that the region between residues 57 and 113 plays a crucial role in oligomerization and membrane insertion. The high binding efficiency of CAMP114-226 suggests this region has great responsibility on membrane binding. The hemolytically inactive fragments showed higher binding efficiency than some of the active fragments. For the hemolytic fragments, higher binding efficiency gave stronger hemolysis. These findings support that CAMP factor has different functional regions for pore-formation.

Streptococcus agalactiae CAMP factor is a protein exotoxin that contains 226 amino acid residues and forms oligomeric pores on susceptible cell membranes and liposomes. In this study, fragments of CAMP factor were created and recombinantly expressed to identify functional domains that are involved in membrane binding, oligomerization, and membrane insertion. Altogether, six truncated forms of CAMP factor were created and assayed. CAMP1-113, CAMP1-170, CAMP57-226, and CAMP171-226 showed different levels of hemolytic activity. CAMP1-56 and CAMP114-226 did not show hemolytic activity or oligomerization ability, but showed binding ability. CAMP114-226 inhibited the hemolytic activity of wild-type CAMP factor, most likely through ‘one-sided’ oligomerization. From the comparison of these fragments, it emerges that the region between residues 57 and 113 plays a crucial role in oligomerization and membrane insertion. The high binding efficiency of CAMP114-226 suggests this region has great responsibility on membrane binding. The hemolytically inactive fragments showed higher binding efficiency than some of the active fragments. For the hemolytic fragments, higher binding efficiency gave stronger hemolysis. These findings support that CAMP factor has different functional regions for pore-formation.