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The role of membrane-intrinsic enzymes of lipid metabolism in complex biological processes is being realized through comprehensive structure function studies. Detailed analysis of substrate-enzyme interactions occurring within the restrictive membrane environment has proved to be exceedingly challenging. Using detergent micelles, we describe a detailed model for substrate recognition and binding by the outer-membrane intrinsic enzyme PagP from Escherichia coli. PagP is an 8-stranded antiparallel β-barrel that transfers a palmitoyl group from a phospholipid molecule to lipid A, the endotoxin component of lipopolysaccharide. This simple modification provides bacterial resistance to host antimicrobial peptides and attenuates the inflammatory response signalled through the host toll-like receptor 4 pathway. We describe a molecular embrasure and a crenel, which display weakened transmembrane β-strand hydrogen bonding, to provide site-specific routes for lateral entry of substrates into the PagP active site. A Tyr147 localized to the L4 loop gates the entry of the phospholipid substrate through the crenel, while lipid A enters via the embrasure. The side chains of the catalytic residues that are located in the extracellular loops point towards the central axis of the enzyme, directly above the active site. An acyl-chain binding pocket known as the hydrocarbon ruler is buried within the transmembrane β-barrel structure, and is optimized to accommodate a 16-carbon saturated palmitate chain. The hydrocarbon ruler, therefore, accounts for PagP's stringent selectivity for a palmitate chain. Substituting Gly88 lining the floor of the hydrocarbon ruler with residues possessing linear, unbranched, aliphatic side chains changes the selectivity of PagP to utilize shorter acyl chains. The serendipitous discovery of an exciton interaction between Trp66 and Tyr26 at the floor of the hydrocarbon ruler provides an intrinsic spectroscopic probe to monitor the methylene unit acyl-chain resolution of PagP. A compromised acyl chain resolution of the Gly88Cys mutant is attributed to an unexpected decrease of the Cys sulfhydryl group pKa within the β-barrel interior, resulting in a burying of a charged thiolate within the PagP core. The structural perturbation associated with the Cys thiolate extinguishes the exciton and expands the acyl-chain selectivity. These molecular details of lateral lipid diffusion and acyl-chain selection provide the first such example for any membrane-intrinsic enzyme of lipid metabolism. / Thesis / Doctor of Philosophy (PhD)

Cataract surgery is currently the most common surgical procedure done in the world. However, within 5 years, approximately half of these patients will develop posterior capsule opacification (PCO). In cataract surgery, the biological lens is replaced with an intraocular lens (IOL). PCO is caused by migration and transformation of residual lens epithelial cells (LEC) that remain in the capsule following the surgery. LECs which have migrated to the posterior capsule within the first month of surgery are thought to be the major contributors to PCO since after this time, the capsule completely seals. A mathematical model has been developed in order to better understand the process of LEC migration during PCO. The model addresses the impact of substrate and substrate modification as well as the presence and absence of the growth factors transforming growth factor beta (TGF(beta)) and fibroblast growth factor (FGF2). It was developed from a first order rate of decay model taken from process control. If the cell speed is divided by the distance travelled by the cell up to the point of posterior capsule breach, the time for the LECs to breach the capsule posterior can be calculated. The model was tested with literature data and was able to predict the effects of cell speed on the presence of various extracellular matrix components and growth factors. It was determined that potentially modification with fibronectin may be useful for the prevention of PCO Preliminary experimental validation of the model was performed by modifying silicone substrates with various extracellular matrix derived peptides. Results demonstrate that peptide modified surfaces may be more resistant to EMT by increasing cell adhesion and decreasing cell migration. Therefore, this LEC migrational model will be a useful tool in the development of superior IOLs and materials. / Thesis / Master of Applied Science (MASc)

Microwave filters are essential components in high frequency communication systems. The features required for these devices are increasing due to the increase in frequency, caused by the saturation of the electromagnetic spectrum. Among these features, the need for low cost devices with a reduced mass and volume and the need to integrate them with the current planar technology are highlighted. In addition, it is interested to have reconfigurable devices, that is, they can adjust their frequency response, replacing the need of multiple devices. So far, metallic waveguide filters have been used, but lately Substrate Intregated Waveguide (SIW) technology has appeared, and within this family, the innovative Empty Substrate Integrated Waveguide (ESIW). ESIW technology is empty, so that, it can be filled with liquid crystal (LC). The LC is a dielectric material with variable permittivity, which makes the filter can change its center frequency and bandwidth. There are several commercial tools based on numerical methods that enable to carry out the analysis and design of these structures, but they require a very high computational time during the analysis process. This affects negatively the automated design of these structures. On the one hand, an efficient and accurate analysis tool is developed in the thesis by following a strategy that consists on dividing the device under analysis in simple building blocks: waveguides filled with dielectric material, change of medium, discontinuities between guides and dielectric discontinuities. All of them are canonical structures or sufficiently simple that can be analyzed with modal methods. The Generalized Scattering Matrix (GSM) of each block is obtained, and they are linked in cascade by using an efficient technique. The accuracy and effectiveness of this tool are checked using it to analyze multiple filters, comparing the result with a commercial software. Furthermore, an analysis about how the variation of the permittivity of the liquid crystal affects in the frequency response of the filter is made. On the other hand, a tool for designing filters for high frequency communications is developed, in order to integrate it into a Computer Aided Design (CAD) tool. To do this, the classical techniques for designing waveguide filters are followed by adapting them to the new topology under design. Different synthesis and optimization strategies are implemented. These strategies are based on the synthesis of a starting point, the segmentation of the structure under design and the hybridization of different optimization algorithms. Also, a tool for calculating the values ​​of the permittivity of the LC that allows the reconfiguration of the filter is developed. These new permittivities enable to obtain a new desired frequency response. This tool has been used to design various microwave filters on H plane, using ESIW technology filled with liquid crystal. The accuracy and the effectiveness of this tool are checked. In addition, the reconfiguration tool designed is evaluated. Finally, a study of the manufacturing aspects is done, such as how to feed the filter and the solutions to polarize the liquid crystal. Taking into account all these considerations a prototype is manufactured and measured.

Increased access to energy is a key factor to reduce poverty and to gain increased development and prosperity. Access to energy is not equally distributed globally. On average a Swedish person consumes more energy than 12 individuals in Tanzania. The Msambara village arose in the 1930s. 80% of the inhabitants are children and the families are large. Life expectancy is 52 years for women and 54 for men. The entire region is very poor and the standard of living in Msambara is low, even compared to other parts of the country. In rural Msambara in Tanzania the cooking method is very primitive and is usually carried out indoors on three stones. Indoor pollution is contributing to eye infections. Incomplete combustion not only emits greenhouse gases increasing global warming, but also more directly emits particles effecting human health. Women and girls are particularly vulnerable to burning caused by air pollution, especially since they are responsible for cooking. The women pull a heavy load in the household work. They are responsible for raising and caring for children, managing farms, collecting firewood, fetching water, visiting the market and cooking for the family. The adoption of biogas technology reduces the need for traditional energy and thereby reduces environmental degradation. In addition, the residue is an improved agricultural fertiliser. Biogas technology has social considerations; burning of biomass for cooking reduces indoor air pollution and reduces workload to collect firewood, often performed by women. Moreover, biogas is desirable from an economic point of view. The method in this work can conveniently be divided into three different parts. These are the designing and construction process as well as the socio-economic study. The socio-economic component is split into two different sections, which are field study and data modelling. The study shows that the social negative impact may or may not be reduced by the introduction of the biogas facility, but the biogas facility in the way it is made will potentially contribute to sustainable economic growth for the household. It also shows that the household’s organic waste produced from human, animal, kitchen and garden waste is enough to provide the necessary gas needed for cooking and no additional firewood will be needed. Two diary cows, in addition to the household’s waste, will provide enough gas needed to introduce cooling capacity for food storage. By adding cooling capacity in the household, time will be saved from the food preparation process but the introduction of biogas itself will not reduce the individual work burden when no cooling capacity is installed. The study also shows that placing the digester in the ground makes a stable environment for the mesophilic and methanogenic microbes. The Socio-economic study indicates that by introducing an alternative method to cooking, positive health effects will arise and the household benefits economically. It is not possible from this thesis to conclude that by adding and treating human toilets as a substrate that possible health benefits will take place. / Sammanfattning Ökad tillgång till energi är en nyckelfaktor för att minska fattigdom och för att få ökad utveckling och välstånd. Tillgången till energi är inte jämnt fördelad globalt. En svensk person konsumerar i genomsnitt mer energi än 12 individer i Tanzania. Byn Msambara uppstod på 1930-talet, 80 % av invånarna utgörs av barn, familjerna är stora. Medellivslängden är 52 år för kvinnor och 54 år för män. Hela regionen präglas av fattigdom och levnadsstandarden i Msambara är låg, även jämfört med andra delar av landet. I Msambara som ligger på Tanzanias landsbygd är tillagningsmetoden mycket primitiv och utförs vanligen inomhus på tre stenar. Den rökiga inomhusmiljön leder ofta till ögoninfektioner. Den ofullständiga förbränningen frigör inte bara växthusgaser och bidrar till den globala uppvärmningen, utan mer direkt avges partiklar som påverkar människors hälsa. Kvinnor och flickor är särskilt utsatta för luftföroreningar eftersom de är ansvariga för matlagning. Kvinnorna drar ett tungt lass i hushållsarbetet. De är ansvarig för uppfostran och vårdnaden utav barnen, hushållsarbetet, sköta jordbruket, samla ved, hämta vatten, besöka marknaden och att laga familjens mat. Introduktion av biogasteknik minskar behovet av traditionell energi och miljöbelastningen. Rötresten är dessutom ett förädlat gödningsmedel. Biogas introduktionen ger positiva sociala konsekvenser då det leder till reducerade luftföroreningar inomhus och minskar behovet av att införskaffa ved, vilket nästan undantagslöst utförs av kvinnor. Dessutom är biogas positivt ur ekonomisk synvinkel. Arbetsmetoden är tydlighetens uppdelat i tre delar. Nämligen dimensionering av rötkammaren, uppförandet av densamma och en okonstlad socioekonomisk studie som i sin tur är uppdelad i en fältstudie och simulering. Studien kan inte visa på att summan av de sociala negativa effekterna minskar med införandet av biogasanläggningen men däremot att den potentiellt bidrar till stärkt ekonomi för hushållet. Den visar också att hushållens organiska avfall som produceras i form av avfall från människor, djur, kök och trädgård är fullt tillräckligt för att producera den nödvändiga gasen som behövs för matlagning. Ingen ytterligare ved kommer att behövas. Två kor förutom hushållets avfall ger den biogas som krävs för att driva ett kylskåp vilket ökar hållbarheten vid matförvaring. Genom tillförandet av kyleffekt till hushållet frigörs tid vilket innebär en tidsbesparing. Införandet av biogas i sig minskar inte den individuella arbetetsbördan. Studien visar också att placeringen av rötkammaren i marken utgör en stabil miljö för de mesofila metanogena. Den socioekonomiska studien visar att införandet av den alternativ matlagningsmetoden medför positiva hälsoeffekter och är ekonomiskt gynnsamt för hushållet. Från denna studie är det inte möjligt att dra slutsatsen att tillförandet och behandlingen av den mänskliga toaletten i rötprocessen ger hälsofördelar.

Glucose is the primary fuel for the brain and also important for exercising muscle. The purpose of the thesis was to investigate the effects of the glycaemic index (GI) of mixed meals on appetite, cognitive performances and metabolic responses during intermittent exercise in recreationally active adults. Study one investigated whether a low GI (LGI) breakfast (GI = 42.5) could suppress appetite and reduce energy intake (EI) of 12 recreationally active females (28.2 ± 8.0 years) more than a high GI (HGI) breakfast (GI = 73.5). Area under the curve of the appetite score (AS AUC) following LGI breakfast was significantly greater than the HGI trial during the 60-min postprandial (pp) period (2568 ± 1027 vs. 2198 ± 821 mm∙min, p = 0.025). The HGI breakfast facilitated a stronger appetite suppressing effect up to eight hours post breakfast than the LGI trial (18834 ± 3906 vs. 21278 ± 3610 mm∙min, p = 0.028). The EI on the LGI trial day was significantly higher than on the pre-trial day (2,215 ± 576 vs. 1,748 ± 464 kcal, corrected p = 0.008). Fourteen recreationally active males (34.5 ± 8.9 years) in study two consumed the LGI (GI = 41.3) and HGI (GI = 74.3) breakfasts in the laboratory and then prescribed LGI and HGI meals in the free living environment. In line with study one, the AS AUC was significantly smaller following HGI than LGI breakfast over the 60-min pp period (2,989 ± 1,390 vs. 3,758 ± 1,290 mm∙min, p = 0.027). The HGI meals (GI = 76.9) suppressed appetite more than the LGI meals (GI = 39.6) over 12 hours on the trial day (35,454 ± 9,730 vs. 41,244 ± 8,829 mm∙min, p = 0.009) although energy balance was not different between trials. Study three investigated whether following a LGI breakfast (GI = 42.2) providing 1 g CHO kg-1 BM could result in a better vigilance and attention than a HGI breakfast (GI = 72.4), and reduced lunch EI in 16 recreationally active males (24.4 ± 3.6 years). A significant trial x time effect in the interference time of the Stroop Colour Word Task (SCWT) (p = 0.039) showed that the LGI breakfast maintained the attentional performance up to 90-min pp. Both high pre-task glucose concentration ([Glucose]) at 15-min pp and low pre-task [Glucose] at 105-min pp in the HGI trial were associated with unfavourable outcomes in vigilance in the Rapid Information Processing Task (RIPT). The LGI pre-task [Glucose] returning back to fasting level at 60-min pp was associated positively with the response time. The pre-lunch AS was a significant predictor of the lunch EI per fat free mass which explained 21% and 26% of variance in the LGI and HGI trials respectively. No significant difference was found in the ad libitum lunch EI between trials. Sixteen recreationally active males (27.8 ± 7.7 years) in study four consumed a LGI (GI = 42) and a HGI breakfast (GI = 72.8) providing 1.2 g CHO kg-1 BM consumed 60 minutes prior to intermittent running on two separate mornings. Better attentional performance at 150-min pp was found following LGI than HGI breakfast. The significant trial x time interaction in the SCWT (p = 0.045) showed the shortest interference time performed after the last exercise session in the LGI trial. The amounts of CHO and fat being oxidized were comparable between trials during three sessions of 16-min intermittent running with an average intensity of 65% V̇O2max. In conclusion, the pre-meal appetite sensation is more predictive of the subsequent meal EI than the pre-meal [Glucose]. The meal strategy for weight management in recreationally active adults may focus on greater appetite suppression by selecting HGI foods whilst maintaining healthy eating guidelines. Recreationally active males performing sports requiring high levels of vigilance and selective attention with low physical activity levels can benefit up to 60–90 min pp from the LGI breakfast. Their attentional performance can benefit from the LGI breakfast with moderate to high intermittent intensities in the late exercise period at 150–min pp. Recreationally active adults should consider the timing of meal consumption in relation to performing intermittent exercise, in order to maximize the advantages from the LGI or HGI breakfasts for cognitive performance or appetite suppression. They may be more liberal in pre-exercise food choices if substrate oxidation during intermittent running is only of their concern.

Bacterial endosymbionts occur in diverse fungi, including members of many lineages of Ascomycota that inhabit living plants. These endosymbiotic bacteria (endohyphal bacteria, EHB) often can be removed from living fungi by antibiotic treatment, providing an opportunity to assess their effects on functional traits of their fungal hosts. We examined the effects of an endohyphal bacterium (Chitinophaga sp., Bacteroidetes) on substrate use by its host, a seed-associated strain of the fungus Fusarium keratoplasticum, by comparing growth between naturally infected and cured fungal strains across 95 carbon sources with a Biolog((R)) phenotypic microarray. Across the majority of substrates (62%), the strain harboring the bacterium significantly outperformed the cured strain as measured by respiration and hyphal density. These substrates included many that are important for plant-and seed fungus interactions, such as D-trehalose, myoinositol, and sucrose, highlighting the potential influence of EHB on the breadth and efficiency of substrate use by an important Fusariurn species. Cases in which the cured strain outperformed the strain harboring the bacterium were observed in only 5% of substrates. We propose that additive or synergistic substrate use by the fungus bacterium pair enhances fungal growth in this association. More generally, alteration of the breadth or efficiency of substrate use by dispensable EHB may change fungal niches in short timeframes, potentially shaping fungal ecology and the outcomes of fungal-host interactions.

The application of the thermally sprayed WC-Co and Cr 3C2-NiCr coatings have been widely used in industry for its superior sliding, abrasive and erosive wear properties. The Al2O3 coating possessing high hardness can be utilized to improve the wear resistance of the metallic surface. Replacing the stamping tool material from steel to lightweight aluminium with protective coatings offers significant gains such as reduction in power costs and increasing operational efficiency during manufacturing of sheet automobile parts. In this study, the WC-CoCr and Cr3C2-NiCr coatings were sprayed with High Velocity Air-Fuel (HVAF) process and Al2O3 coating was deposited using Atmosphere Plasma Spraying (APS) process. The coatings were evaluated and compared based on the results of roughness tests, hardness tests, adhesion tests, quantitative imaging analysis and microstructure analysis. The experimental results revealed that the roughness value varied in a lower range which implies of a high density of the coatings. It was found that coatings have an extremely high hardness value as tested by the Vickers hardness test. The WC-Co coating was found to be the hardest (1215.2HV). The adhesion test was implemented according to the ASTM C633-79 standard. The result showed that WC-Co and Cr3C2-NiCr coatings without a bond coat had high tensile strength, higher than the respective glue strength (63.54MPa, 75.89MPa). Whereas, the Al2O3 coating has a much lower tensile strength (15.2MPa, 25.68MPa, with and without bond) than others and using a bond coat layer does not contribute to an increase in adhesion strength of the cermet coatings. The coating thickness was evaluated using Light Optical Microscopy (LOM) and microstructure analysis was carried out using Scanning Electron Microscopy (SEM). From the microstructure analysis, it was observed that all the coatings have a dense microstructure, very low porosity and low oxide inclusions in top coat. The WC and Cr3C2 grains retain a large volume fraction of finely dispersed in matrix. The alumina coating was found to have low levels of un-melted or the re-solidified particles in the coating. All of the aforementioned analysis and results reveal that WC/CoCr and Cr3C2-NiCr coating show promising potential for press tool applications. However, abrasive resistance test still remained to be done and will be performed in the future. The result of it can reveal the actual wear resistance between two coatings in reality and will be helpful in determining the better protective coating for aluminium press tools

Temperature dependence of marine plankton respiration is an important factor in understanding the function and changes in the ecosystem of the ocean. The aim of this study is to test the temperature sensitivity (Q10) of plankton respiration. The oxygen optode method was used to measure plankton respiration. Natural water samples from the Baltic Sea was incubated at short (in situ +1, +2, +3°C) and long (in situ +5, +10, +20°C) temperature intervals with influence of dissolved organic matter (DOC). The Arrhenius equation and Q10-model was used to determine the temperature dependence (Q10) of respiration at different temperatures. There was a significant difference in Q10 between short temperature intervals at low temperatures (p=0,008) and long temperature intervals at higher temperatures. There was no significant difference between long and short temperature intervals when DOC was added (p=0,094). A significant effect could be seen with the DOC enrichment at low temperatures, where the Q10-values became significantly lower (p=0,002) after DOC addition. This effect could, however, not be seen at higher temperatures (p=0,117). Together with results from earlier studies it was concluded that the difference in temperature depends on the actual temperature and not the length of the interval. Lowered temperature dependence at raised DOC concentration, was the opposite of what was expected. The results suggest that the importance of temperature for CO2 emissions and development of hypoxia in the sea may have been underestimated.

Biocatalysis is an ever evolving field that uses enzymes or microorganisms for chemical synthesis. By utilizing enzymes that generally have evolved for specific reactions under mild conditions and temperatures, biocatalysis can be a more environmentally friendly option compared to traditional chemistry. Amide-type chemistries are important and bond formation avoiding poor atom economy is of high priority in organic chemistry. Biocatalysis could potentially be a solution but restricted substrate scope is a limitation. Esterases/lipases usually display broad substrate scope and catalytic promiscuity but are poor at hydrolyzing amides compared to amidases/proteases. The difference between the two enzyme classes is hypothesized to reside in one key hydrogen bond present in amidases, which facilitates the transition state for nitrogen inversion during catalysis. In this thesis the work has been focused on introducing a stabilizing hydrogen bond acceptor in esterases, mimicking that found in amidases, to develop better enzymatic catalysts for amide-based chemistries. By two strategies, side-chain or water interaction, variants were created in three esterases that displayed up to 210-times increased relative amidase specificity compared to the wild type. The best variant displayed reduced activation enthalpy corresponding to a weak hydrogen bond. The results show an estimated lower limit on how much the hydrogen bond can be worth to catalysis. MsAcT catalyze kinetically controlled N-acylations in water. An enzymatic one-pot one-step cascade was developed for the formation of amides from aldehydes in water that gave 97% conversion. In addition, engineered variants of MsAcT with increased substrate scope could synthesize an amide in water with 81% conversion, where the wild type gave no conversion. Moreover, variants of MsAcT displayed up to 32-fold change in specificity towards amide synthesis and a switch in reaction preference favoring amide over ester synthesis. / <p>QC 20161125</p>