Of the organisms with sequenced genomes, plants appear to possess the most rhomboid protease-encoding genes. However, our knowledge of processes in plants that involve Regulated Intramembrane Proteolysis (RIP) and rhomboid proteases remains low. As expressed recently by other researchers, finding a natural substrate for a rhomboid protease represents the biggest experimental challenge. Using yeast mitochondria-based assays, a potential link between the plastid translocon component Tic40 and organellar rhomboid proteases was recently uncovered. In this particular link, rhomboid proteases appear capable of influencing the pattern of imported Tic40 in yeast mitochondria. Tic40 may thus represent a natural plant target of organellar rhomboid proteases. Here, we obtained further motif-oriented evidence supporting Tic40 as a natural plant rhomboid substrate. A comparative analysis of sequences revealed that Tic40 may also possess similar TMD motifs found in the model substrate, Spitz. Rhomboid proteases often require these motifs to cleave substrates within intramembrane environments. Using site-directed mutagenesis and yeast mitochondria assays, the impact of mutations occurring in the motifs ASISS, GV, QP, and GVGVG of Tic40 was assessed. In terms of cleavage and changing the pattern of imported Tic40, some of the mutations showed decreased activities and a few showed enhancements. More importantly, the overall observed pattern associated with select Tic40 mutations resembled the characteristics reported for the model substrates. In particular, mutations in the Tic40 GV motif produced similar results as that observed with Spitz, by drastically decreasing or increasing cleavage as a function of amino acid sequence. / Thesis (Master, Biology) -- Queen's University, 2010-05-30 10:22:07.72

The mixed sand and gravel barrier beaches located on the South Island's East Coast are formed predominantly of Greywacke, eroded from the mountains, and transported via the major river systems. These barriers act as the interface between the South Pacific Ocean and the surrounding hinterland. In times of high energy coastal events, breaching is common. This thesis examines the form and function of the Waihao-Wainono barrier, a section of the coastline situated north of the Waitaki River. Breaches along this part of the barrier are frequent and several have rendered the surrounding farmland unusable for several years due to the effects of saltwater inundation. There is some concern among the local community as to exactly why the barrier breaches at certain locations and not others, making land planning and management a difficult task for farmers. Several of the local landowners believe that since the construction of the Waitaki Dam in 1935, a significant decrease in sediment size along the barrier has occurred. It is also thought that the barrier form has experienced substantial change. Through the use of physical techniques used in the field of coastal science, 17 sites along the Waihao-Wainono barrier were studied. Excavations were carried out, surface and substrate profiles recorded and sediment samples collected from the surface. sub-surface and substrate of the barrier. Analysis of the barrier form and barrier volume concluded that the past breach sites consisted of steeper lower foreshore slopes than the non-breach sites, and at two sites, the substrate was not reached. Breach areas display the greatest barrier volume of all the study sites, which is contrary to belief. In relation to the surface sediments, the majority of barrier profiles displayed the distinct mean grain size cross shore zonation, characteristic of mixed sand and gravel beaches. The best and most consistent surface sorting was also identified as being a characteristic of the breach sites. The sediment size is not shown to have drastically reduced over the thirty year sampling period as was perceived by the local community. Within the sub-surface of the barrier, the sediments displayed chaotic sizes and generally poorly sorted material. Several of the breach sites contained a distinct change in sediment size between the coarser surface layer and the finer layer located immediately below. This layering of coarse and fine sized sediments leads to differences in permeability within the barrier, which is thought to be a major factor in why these sites have breached. Resulting from these findings, a group of characteristics of breach sites was formed and several predictions made as to where the barrier may breach in the near future.

This thesis explores the roles of physical and mathematical modelling in the prediction of temperature profiles in the composting process. A literature-based evaluation of the performance of laboratory- and pilot scale composting reactors, showed that physical models used in composting research frequently do not properly simulate the full-scale composting environment, and may therefore produce results which are not applicable at full scale. In particular, self-heating, laboratory-scale, reactors typically involve significant convective/conductive/radiative losses, even with insulation present. This problem can be overcome by using controlled temperature difference or controlled heat flux laboratory reactors, which allow convective/conductive/radiative heat fluxes to be controlled to levels close to those occurring in full-scale systems. A new method of assessing the simulation performance of composting systems is presented. This utilises the areas bounded by the temperature-time profile and reference temperatures of 40 and 55 ℃ (A₄₀ and A₅₅), the times for which these temperatures are exceeded (t₄₀ and t₅₅), and times to peak temperature. An evaluation of published temperature profiles showed a marked difference in these parameters when comparing many laboratory- and full-scale reactors. The impact of aeration is illustrated, and laboratory- and pilot-scale reactors able to provide good temperature profile simulation, both qualitatively and quantitatively, are identified. Mathematical models of the composting process are reviewed and their ability to predict temperature profiles assessed. The most successful models in predicting temperature profiles have incorporated either empirical kinetic expressions, or utilised a first-order model, with empirical corrections for temperature and moisture. However, no temperature models have been able to predict maximum, average and peak temperatures to within 5, 2 and 2 ℃ respectively, or to predict the times to reach peak temperatures to within 8 h, although many models were able to successfully predict temperature profile shape characteristics. An evaluation of published constant-temperature and varying-temperature substrate degradation profiles revealed very limited evidence to support the application of single exponential, double exponential or non-logarithmic Gompertz functions in modelling substrate degradation kinetics, and this was identified as a potential weakness in the temperature prediction model. A new procedure for correcting substrate degradation profiles generated at varying temperature to a constant temperature of 40 ℃ was developed and applied in this analysis, and on experimental data generated in the present work. A new approach to the estimation of substrate degradation profiles in the composting process, based on a re-arrangement of the heat balance around a reactor, was developed, and implemented with both a simulated data set, and data from composting experiments conducted in a laboratory-scale constant temperature difference (CTD) reactor. A new simulated composting feedstock for use in these experiments was prepared from ostrich feed pellets, office paper, finished compost and woodchips. The new modelling approach successfully predicted the generic shape of experimental substrate degradation profiles obtained from CO2 measurements, but under the conditions and assumptions of the experiment, the profiles were quantitatively different. Both measured CO2-carbon (CO2-C) and predicted biodegradable volatile solids carbon (BVS-C) profiles were moderately to well fitted by single exponential functions with similar rate coefficients. When corrected to a constant temperature of 40 ℃, these profiles gave either multi-phase or double exponential profiles, depending upon the cardinal temperatures used in the temperature correction procedure. If it is assumed that the double exponential model generated is correct, this work provides strong evidence that a substrate degradation curve generated under appropriate laboratory conditions at 40 ℃ would, given the correct cardinal temperatures, generate a correct substrate degradation profile under varying temperature conditions, and that this in turn would enable an accurate and precise prediction of the temperature profile using a heat and mass balance approach. This finding opens the door for the development of a simple laboratory test for composting raw material characterisation, but underlines the need for accurate estimates of the physical cardinal temperatures. Experimental factors appear to be the likely cause of the dysfunction between previously reported substrate degradation patterns and existing substrate degradation models, and suggestions for further research are provided in order to more precisely and accurately quantify these factors.

Although a large number of works on nanoimprint lithography (NIL) techniques have been reported, the the ability for three-dimensional (3-D) patterning using NIL has not been fully addressed in terms of the mold fabrication and imprint processes. Patterning 3-D and multilevel features are important because they eliminate multiple steps and complex interlevel alignments in the nanofabrication process. The 3-D and multilevel mold design and fabrication, and imprint processes have been studied and investigated in this research work. In the UV-NIL technique, a transparent mold with micro/nanostructure patterns on its surface is allowed to be replicated on UV curable polymer without the need of high applied pressure or temperature. UV-NIL has the potential to fabricate micro/nanostructures with high resolution, high reproducibility, low cost, high throughput and is capable of 3-D patterning. This research focuses on two aspects; the development of mold making and imprint processes. In the process of making a master mold, an EBL technique was employed for writing patterns on e-beam resists. PMMA positive resist was used for 2-D patterning and ma-N2403 negative resist from Microresist Technology was used for 3-D patterning. After being developed, the 3-D mold pattern was transferred onto quartz substrate using a single-step reactive ion etching (RIE) technique. A number of challenging issues such as surface charging, electron scattering and proximity effects surfaced during the EBL pattern writing on insulating and transparent molds. A number of new approaches have been developed for suppressing the charging effects in the 2-D and 3-D patterning. Using thin metallic coating on the quartz substrates or on top of the resist, or conductive polymer coating using PEDOT/PSS on top of the resist has demonstrated excellent results in a 2-D structure with a high aspect-ratio of 1:10 and feature sizes down to 60 nm. In 3-D patterning, two approaches have been followed; the critical energy method and/or a top coating of conductive polymer (PEDOT/PSS) layer. Isolated 3-D structures with feature sizes down to 500 nm were successfully fabricated using the first method while by using the second method, dense 3-D structures patterns with feature sizes down to 300 nm, on 400 nm pitch have been demonstrated. In UV-NIL, the surface roughness Rq(rms) should be less than 5 nm, which is important for replicating optical structures and devices. In this work, the RIE process been optimized to yield 2 nm roughness on a patterned quartz surface. This was achieved by optimizing the RIE process pressure of below 6 mTorr. The other part of this thesis is on replication or imprinting of 2-D and 3-D structures. In the process of replicating the master mold profiles, the imprint processes were carried out using a vacuum operated manual imprint tool which was attached to a Mask Aligner UV illumination system. In 2-D imprinting, resist sticking on the vertical side wall was the main issue, especially on high aspect ratio structures. Meanwhile in 3-D imprinting, the imprint results have shown good reproducibility in up to 15 imprint cycles, where the issue of Ormocomp soft/daughter mold cracking after long UV exposure had limited the repetition of the imprint cycles. In this thesis, the 2-D and 3-D resist patterning on insulating substrates using the EBL technique have been demonstrated with the assistance of a number of developed charge suppression methods. Single-step RIE pattern transfer onto quartz substrates with surface roughness below 5nm has been achieved. Replication of 3-D and multilevel structures reliably make the UV-NIL technique suitable for future applications such as surface texturing, optical devices and many other complex structures including MEMS.

RNase P is a ubiquitous ribonuclease responsible for removing the 5’ leader of tRNA precursor. Bacterial RNase P contains one RNA (RPR) and one protein (RPP) subunit. However, the number of protein variants depends on the origin. The RNA subunit is the catalytic subunit that in vitro cleaves its substrate with and without the protein subunit. Therefore RNase P is a ribozyme. However, the protein subunit is indispensable in vivo. The objective of this thesis was to understand the mechanism of and substrate interaction in RPR-mediated cleavage, in particular the contributions of the two domains of RPR and the roles of the base at the -1 residue in the substrate. As model systems I have used bacterial (Eco) and archaeal (Pfu) RPRs. The TSL (T-stem-loop) region of a tRNA precursor and the TBS (TSL-binding site) in the RPR S-domain interact upon RPR-substrate complex conformation. A productive TSL/TBS-interaction affects events at the cleavage site by influencing the positioning of chemical groups and/ or Mg2+ such that efficient and correct cleavage occurs consistent with an induced fit mechanism. With respect to events at the cleavage site, my data show that the identity of the residue immediately upstream the 5’ of the cleavage site (at -1) plays a significant role for efficient and accurate cleavage although its presence is not essential. My data also show that the RPR C-domain can cleave without the S-domain. However, the presence of the S-domain increases the efficiency of cleavage but lowers the accuracy. The structure of the S-domain of Pfu RPR differs from that of Eco RPR and my data suggest that the Pfu S-domain does not affect the accuracy in the same way as for Eco RPR. It also appears that the proteins that bind to the Pfu S-domain play a role in formation of a productive TSL/TBS-interaction. It is therefore possible that the proteins of Pfu RNase P have evolved to take over the role of the S-domain with respect to the interaction with the TSL-region of the substrate.

The mixed sand and gravel barrier beaches located on the South Island's East Coast are formed predominantly of Greywacke, eroded from the mountains, and transported via the major river systems. These barriers act as the interface between the South Pacific Ocean and the surrounding hinterland. In times of high energy coastal events, breaching is common. This thesis examines the form and function of the Waihao-Wainono barrier, a section of the coastline situated north of the Waitaki River. Breaches along this part of the barrier are frequent and several have rendered the surrounding farmland unusable for several years due to the effects of saltwater inundation. There is some concern among the local community as to exactly why the barrier breaches at certain locations and not others, making land planning and management a difficult task for farmers. Several of the local landowners believe that since the construction of the Waitaki Dam in 1935, a significant decrease in sediment size along the barrier has occurred. It is also thought that the barrier form has experienced substantial change. Through the use of physical techniques used in the field of coastal science, 17 sites along the Waihao-Wainono barrier were studied. Excavations were carried out, surface and substrate profiles recorded and sediment samples collected from the surface. sub-surface and substrate of the barrier. Analysis of the barrier form and barrier volume concluded that the past breach sites consisted of steeper lower foreshore slopes than the non-breach sites, and at two sites, the substrate was not reached. Breach areas display the greatest barrier volume of all the study sites, which is contrary to belief. In relation to the surface sediments, the majority of barrier profiles displayed the distinct mean grain size cross shore zonation, characteristic of mixed sand and gravel beaches. The best and most consistent surface sorting was also identified as being a characteristic of the breach sites. The sediment size is not shown to have drastically reduced over the thirty year sampling period as was perceived by the local community. Within the sub-surface of the barrier, the sediments displayed chaotic sizes and generally poorly sorted material. Several of the breach sites contained a distinct change in sediment size between the coarser surface layer and the finer layer located immediately below. This layering of coarse and fine sized sediments leads to differences in permeability within the barrier, which is thought to be a major factor in why these sites have breached. Resulting from these findings, a group of characteristics of breach sites was formed and several predictions made as to where the barrier may breach in the near future.

Estuarine environments are threatened by the synergistic effects of anthropogenic disturbance and bioinvasion. The construction of artificial structures (such as pilings and pontoons) provides a habitat resource in close proximity to vessel hulls that may be carrying a wide range of non-indigenous fouling species. In addition, the release and accumulation of toxicants from antifouling (AF) paints on vessel hulls creates a chemical disturbance that may reduce the invasion resistance of native communities. This thesis examines how shipping-related disturbances affect sessile communities, and in particular what role AF paints and artificial structures play in the invasion of estuaries. Using a series of field-based experiments, I found that copper and tributyltin have the potential to influence both the transport of species to a new region, via the application of AF paints on vessel hulls, and their subsequent establishment, via the accumulation of AF biocides in estuaries. Temperature, pH, salinity and turbidity were also related to species distributions. During subtidal surveys of artificial and natural structures I found more non-indigenous species (NIS) on pilings and pontoons than on rocky reef, and shallow floating structures were identified as hotspots for invaders. These findings suggest that artificial structures play an important role in the initial establishment of sessile non-indigenous species in new regions. A subset of NIS were also present on the reefs sampled during the survey and I conducted manipulative experiments to determine factors affecting the invasibility of turf and canopy-forming algal assemblages. The resident assemblage provided a barrier to most invaders, particularly when light and sedimentation levels were also high (i.e. on horizontal substrate). My results suggest that the areas of reef most susceptible to invasion are vertical rock walls and those subjected to disturbances that release space. In summary, this is the first study to relate copper and tributyltin contamination in the field to NIS distributions. My research has also highlighted the potential role of artificial structures in facilitating the establishment of NIS in estuaries and identified invasion threats to rock wall communities and disturbed reefs in estuarine systems.

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 6 uppsatser.

Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 87-92). Also available on the World Wide Web.