Doctoral Theses / Doktorsavhandlingar Institutionen för kemi
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Item Fluxes and transformation of carbon in the Siberian shelf seas under changing environment(2012-05-16) Wåhlström, IréneThe Arctic is especially vulnerable to the increased air temperature caused by emissions of greenhouse gases to the atmosphere, carbon dioxide being one of them. In this thesis, both fieldwork and modelling of the East Siberian Arctic Shelf (the Laptev Sea and the East Siberian Sea) have been carried out to investigate the carbon system in this region. Fieldwork in 2008 revealed two distinct hydrological regimes in the East Siberian Sea, one in the western area and one in the eastern area. The western area is dominated by freshwater from rivers flowing into the East Siberian Sea but also from the Lena River plume coming from the Laptev Sea. Nearly all waters in this area are supersaturated with respect to carbon dioxide compared to the atmosphere due to mineralization of substantial amounts of terrestrial organic matter coming from thawing permafrost and coastal erosion. This excess carbon dioxide may be a potential source to the atmosphere and thus increase the atmospheric greenhouse gas content, a positive feedback mechanism. The eastern area is dominated by inflow of Pacific-derived waters that are clear, salty and nutrient rich and therefore favour primary productivity. Phytoplankton consumes carbon dioxide that lowers its partial pressure (pCO2) making it undersaturated compared to the atmosphere and the Eastern East Siberian Sea becomes a sink for atmospheric carbon dioxide. In addition, the Laptev Sea had supersaturated pCO2 equal to an excess of dissolved inorganic carbon of around ~5 1012 gC, which is in the same order as for the Western East Siberian Sea. This excess is also a potential source of carbon dioxide to the atmosphere that could enhance climate change. Modelling work with a one-dimensional, time dependent coupled physical biogeochemical model confirms this conclusion for the late summer when the pCO2 in the seawater increases due to mineralization and water mixing. Model simulations for the Laptev Sea were utilized to investigate the net annual sea-air flux caused by different forcings; doubled atmospheric partial pressure of carbon dioxide; 4oC air temperature increase; doubling the concentration in the runoff of dissolved organic carbon or nutrients; increasing the river discharge by 25 %; increasing the wind speed by 10 % or a combination of these forcings. The result revealed decreasing uptake of carbon dioxide when changing the river properties except for the increase of nutrients when the uptake of carbon dioxide increased. The uptake also increased with the changed forcings in air temperature, wind speed and atmospheric partial pressure of carbon dioxide, separately and in combination.Item Hydrolytic Synthesis and Physicochemical Properties of TiO2 Nanoparticles: Fundamentals and Applications(2012-04-13) Perez Holmberg, JennyTitanium dioxide (TiO2) nanoparticles were synthesized via the hydrolysis of TiCl4 in order to produce clean, surfactant-free oxide surfaces. By controlling the synthesis and aging conditions, stable particles with well-defined size distributions were obtained. The particles were characterized using dynamic light scattering (DLS), electrospray-scanning mobility particle sizer (ES-SMPS), transmission electron microscopy (TEM), and X-ray diffraction (XRD) in order to gain information about the size, morphology and crystalline structure of the material. Dialysis and storage of the particles at 0°C, 5°C and room temperature gave rise to average particle sizes of around 8, 10, and 18 nm after the first 3 weeks of aging. The sizes obtained from DLS and ES-SMPS were generally in good agreement. Analysis by TEM and XRD shows that the synthesis products are composed of primary particles, about 4 nm in size, predominantly of anatase structure. A small amount of brookite (<10%) is produced. Thermodynamic calculations and experimental data reveal that the formation of the oxide particle proceeds from the condensation of titanium complexes, the first hydrolysis products, from solution. At low pH, a repeated condensation/dissolution process occurs, until an amorphous Ti-oxyhydroxide is formed, which eventually becomes crystalline TiO2. Experimental results from ES-SMPS and DLS indicate that the particles grow to their average measured size by the formation of stable aggregates. The effects of polydispersity on the collective diffusion detected by DLS were examined in detail, and a binary sphere model was proposed, to explain the nonlinear concentration dependence of the diffusion coefficient. The interactions between small particles and larger porous aggregates give rise to nonlinearity in transport behaviour at low volume fractions. A closer look at the surface charging of the particles was done by theoretical calculations and experiments, with indications of size-dependent behaviour. The influence of the TiO2 nanoparticle properties on selected applications was illustrated, using the characteristics of the porous nanoparticle surface on Ti dental implants and the interactions of free TiO2 nanoparticles with Ca2+ rich lipid bilayers. The findings of these studies validate that the direct exposure of the oxide surface to the surrounding electrolyte is significant in terms of understanding interfacial processes from the biological perspective.Item MECHANISTIC INVESTIGATIONS OF TRANSITION METAL CATALYZED REACTIONS(2011-12-14) Kleimark, JonatanTransition metal catalyzed reactions have had a large impact on the human progress for the last century. Several extremely important areas, such as the agricultural industry and the plastic industry, have benefited from this development. The evolution of different transition metal catalysts has also been very important for the pharmaceutical industry. One vital factor when developing new and more effective catalysts is to obtain mechanistic insights. In this thesis, several different methods to investigate mechanisms for transition metal catalyzed reactions are presented. The factors controlling regioselectivity for a palladium catalyzed allylic alkylation has been studied. Pre-formed (η3-allyl)Pd complexes were used to minimize dynamic processes. In the study it was found that the regioselectivity depends mainly on steric interactions, rather than electronic effects. For complexes with less steric hindrance, the trans effect controlls the selectivity. Furthermore, the mechanism for a sulfinyl nucleophile, employed in the same type of reaction, has been studied and the mode of attack has been revealed. The importance of a fast palladium catalyzed Mislow-Braverman-Evans rearrangement to ensure that the correct product was formed, was also disclosed. The important Mizoroki-Heck reaction has been investigated in two different studies. The first study revealed the mechanistic pathway for a Pd(II) catalyzed domino Mizoroki-Heck-Suzuki diarylation reaction. The dependence of benzoquinone as the re-oxidant, in order to achieve the diarylation product, was explained by its ability to coordinate to the palladium moiety, thereby allowing access to a new low-energy pathway to the product. In the second study, a new and mild nickel catalyzed variant of the Mizoroki-Heck reaction was presented and the mechanistic pathway for the reaction was introduced. In addition to this, the reasons for several unsuccessful conditions and additives were uncovered. The development of new, environmentally more benign, catalysts for cross coupling reactions is important. Iron is one of the most promising metals for this purpose, but the mechanistic knowledge of this reaction is still not comprehensive. In this thesis, several mechanistic and computational studies reveal new insights into this reaction, paving the way to develop new and more effective catalysts and conditions for the reaction.Item Chiral Lithium N,P-amide Complexes. Synthesis, Applications and Structural Studies by NMR and DFT.(2011-11-28) Rönnholm, PetraEnantiospecific synthesis reactions are of intense interest, owing to the increasing request for enantiopure compounds in both research and industry. Lithium amides containing a secondary chelating group are a class of powerful ligands for asymmetric addition reactions. Based on earlier experiences with lithium N,O and N,S amides, synthesis and properties of chiral lithium N,P amides and their use in asymmetric addition are investigated in the present thesis. Several chiral amines were synthesized with previously published methods, which were improved in different ways. A new synthetic route towards chiral aminophosphines via cyclic sulfamidates has been developed. The use of silica in the synthesis of sulfamidate and the chiral aminophosphine shortened the reaction time considerably, compared to previous methods. The reactions are fast, clean and highyielding. Furthermore, the synthesis could successfully be scaled up with no loss in yield or purity and gives a general and simple route to a wide variety of chiral N,P-ligands from cheap and readily available amino acids. Solution studies using low temperature 6Li-NMR showed that the chiral lithium N,Pamides form various types of dimers depending on solvent and substituents in the amino acid backbone. The Li-P interactions in these complexes proved much stronger than expected, as indicated by the 6Li-31P coupling constant. A stability study on the aminophosphines with 31P-NMR proved they are relatively air stable. The newly synthesized lithium N,P amides were used as ligands in the asymmetric 1,2-addition of n-BuLi to benzaldehyde. The chiral N,P-ligands were found to induce asymmetry to similar or better extent, compared to previously reported chiral N,Oand N,S-ligands. Enantiomeric ratios up to 98:1 were obtained at –116 °C. The experiments were complemented by quantum-chemical calculations employing Density-Functional Theory and Molecular Mechanics (MM), in order to rationalize the experimental findings. For the MM calculations, a tailored force field was developed to allow a proper description of the Li-N interaction. Both the aggregation and solvation of the ligand and the reaction mechanism were investigated. The predicted solvation and aggregation states as well as the enantioselectivities were in good accordance with experiment, provided that dispersion interaction was taken into account in a proper way. It was found that Li-π and π-α-H interactions and solvation within the complexes are the major contributions to the energy differences between the more stable (R)-transition state compared to its corresponding (S)-transition state.Item Influence of resuspension on sediment-water solute exchange and particle transport in marine environments(2011-11-24) Almroth Rosell, ElinMarine sediments contain a large pool of nutrients, which if released would contribute to increased eutrophication, in spite of decreased nutrient loads from land and atmosphere. Resuspension is a process, which might influence the release of nutrients from the sediment to the overlying water. The influence of resuspension on benthic fluxes of oxygen, dissolved inorganic carbon (DIC), nutrients, dissolved iron (dFe) and dissolved manganese (dMn) was therefore investigated in three different marine environments. The measurements were performed using a benthic lander with the advantage of operating in situ. The method of measuring the effects of resuspension was developed in the archipelago of Gothenburg (Paper I). This method was then further improved and used during field studies in the Gulf of Finland (GoF; Paper II) and in a Scottish sea loch (Paper III). During the latter study also the effects of massive (simulating dredging or trawling) and repeated resuspension events on the benthic fluxes were studied. Natural resuspension significantly increased the oxygen consumption in the GoF and at a station with organic rich sediment in Scotland. There were no significant effects of natural resuspension on nutrient, DIC and dMn fluxes, but the fluxes and concentrations of dFe increased at stations with low bottom water oxygen concentrations (GoF). Massive resuspension increased the oxygen consumption enormously and instantly changed the bottom water concentrations of phosphate (which decreased), DIC, silicate and ammonium (which increased). Results confirmed that the general magnitude of phosphate fluxes was dependent on the oxygen regime (GoF; Paper IV). However, results also showed a strong correlation between phosphate and DIC fluxes during anoxic conditions implying that phosphate fluxes are controlled by input and degradation of organic matter under anoxia. The internal load was calculated to be about 66 000 ton P yr-1 in the GoF. If all oxic bottoms below 40 m would turn anoxic the internal load was computed to increase with about 35 000 ton P yr-1. Results from a fully coupled high-resolution biogeochemical-physical ocean model, including an empirical wave model, showed that a large fraction of the sedimentary organic carbon has at least once been resuspended, and the largest contribution of resuspended organic matter to the total transport of particulate organic matter occurred at shallow transport and erosion bottoms (long-term average, 1979-2007) in the Baltic Sea (Paper V). The fraction of resuspended organic matter in the deepest areas of the Baltic Sea was low (< 10%) even though there was a large horizontal transport of suspended organic matter and a high sedimentary content of it. A map of different bottom types, accumulation, transport and erosion bottoms, was also created.Item New insights in contact allergy and drug delivery. A study of formulation effects and hapten targets in skin using two-photon fluorescence microscopy(2011-11-08) Simonsson, CarlThe skin is a remarkable barrier, protecting us from invasion of e.g. harmful microorganisms and UV-radiation. However, the skin is not adopted to resist repeated exposure to the multitude of xenobiotics introduced into modern society. Some of these chemicals are skin sensitizers, and exposure can lead to the development of contact allergy. Contact allergy has significant social and economic consequences, both for the individual and for society. It is therefore important to prevent sensitization. The skin also constitutes a potential route for administration of drugs, and much effort is put into the development of cutaneous and transdermal drug delivery systems. The work of this thesis aims to improve the understanding of processes related to the interactions between the skin and topically applied compounds, i.e. drugs and skin sensitizers. Specifically, two-photon microscopy has been used to study the cutaneous absorption and distribution of model drugs and a series of model skin sensitizers. Improved cutaneous absorption was demonstrated using formulations composed of lipid cubic phases. The work also showed elevated sensitization potency of haptens depending on delivery vehicles. Putative mechanistic explanations for the observed effects have been proposed. Specifically, phthalates were shown to increase the sensitization potency of isothiocyanates. The phthalate-induced effect could be linked to a PSU-targeted delivery of the haptens into the skin. It could also be shown that vehicles alter hapten reactivity to stratum corneum proteins leading to variations in sensitization potency. Moreover, hapten protein targets in skin have been identified using caged fluorescent model hapten. Specifically, basal cell keratinocytes and the keratins were identified as specific hapten targets in the skin. In conclusion, the work presented in this thesis contributes to the general understanding of the mechanisms involved in the cutaneous absorption of topically applied drugs and skin sensitizers. It also demonstrates the capabilities of using TPM when investigating the interactions between the skin and xenobiotics.Item Conformational Dynamics of Rhodopsins Visualized by Time-resolved Wide Angle X-ray Scattering(2011-10-27) Malmerberg, ErikRhodopsins are a family of light-sensitive proteins found in the cellular membranes of a wide range of living organisms. These membrane proteins share a common molecular architecture and are able to use light energy to perform a variety of different biological functions. Mapping the conformational changes required for these proteins to function is important for understanding how light energy is used for energy transduction and sensory perception in biological systems. In order to visualize these conformational changes over time, the emerging technique of time-resolved wide angle X-ray scattering (TR-WAXS) was employed. Several technical and analytical developments of this solution based method were made during the course of this work, including the development of a new data collection strategy based on a rapid readout X-ray detector. The light-driven proton pumps bacteriorhodopsin and proteorhodopsin were the first membrane proteins to be characterized using TR-WAXS. The results from these studies indicated that significant α-helical rearrangements precede the primary proton transfer event in bacteriorhodopsin. Comparison with the evolutionary related proteorhodopsin provided important insights into shared conformational dynamics between the two proton-pumps. Proteorhodopsin was further investigated by probing the conformational changes occurring within its chromophore binding pocket, where the chromophore of proteorhodopsin was substituted with a chemically modified retinal analogue. Comparison between the native and modified form of proteorhodopsin indicated significant chromophore dependant differences in their conformational kinetics. These differences provided new insights into the coupling between retinal isomerisation and protein conformational changes. The conformational dynamics within visual rhodopsin, the primary light sensor of vertebrate vision, were also investigated using TR-WAXS. By using the rapid readout X-ray detector we were able to follow the activation of this G-protein coupled receptor in real-time. Structural analysis further indicated that dramatic conformational changes are associated with the activation of this receptor.Item Physical properties and processes of secondary organic aerosol and its constituents(2011-10-27) Salo, KentAtmospheric aerosol particles are involved in several important processes including the formation of clouds and precipitation. A considerable fraction of the ambient aerosol mass consists of organic compounds of both primary and secondary origin. These organic compounds are often semi-volatile and susceptible to oxidation which makes the organic aerosol a dynamic system, both chemically and physically. Once an aerosol is formed or released into the atmosphere, several processes will begin to alter its chemical and physical properties. The focus of the work presented in this thesis has been to use experimental methods to characterise single aerosol components and more complex experimental systems, involving the formation and processing of secondary organic aerosol (SOA). The volatility of aerosol particles, e.g. the evaporation rate of the particles upon heating, can provide information of several important properties. From an aerosol consisting only of one pure compound it is possible to derive physical quantities like saturation vapour pressure and enthalpy of evaporation. In more complex systems like a secondary organic aerosol the volatility can give information about changes in composition, state of oxidation and degree of internal or external mixing. With the use of a volatility tandem differential mobility analyser (VTDMA), the saturation vapour pressures and enthalpies of evaporation have been determined for pure compounds that are known constituents of ambient aerosol samples i.e. nine carboxylic acids. Two of them were cyclic, pinic and pinonic acid and seven of them were straight chain dicarboxylic acids with number of carbons ranging from C4 to C10. These properties were in addition evaluated for the aminium nitrates of mono-, di-, and trimethylamine, ethyl- and monoethanolamine. The calculated saturation vapour pressures for the carboxylic acids were in the range of 10-6 to 10-3 Pa and the determined enthalpies of evaporation ranged from 83 to 161 kJ mol-1. The corresponding values for the aminium nitrates were for the calculated saturation vapour pressures approximately 10-4 Pa and for the enthalpies of evaporation 54 to 72 kJ mol-1. The VTDMA system has also been utilised to characterise SOA formed in the AIDA and SAPHIRE smog chambers from the ozonolysis of α-pinene and limonene and the change in the SOA thermal properties during OH radical induced ageing. Further, the effect of elevated ozone concentration and radical chemistry on SOA formed from limonene ozonolysis in the G-FROST laminar flow reactor was investigated. In addition, to compare with vapour pressures of aminium nitrates SOA generated from photooxodation of alkyl amines have been characterised in the EUPHORE smog chamber. The calculated vapour pressures of all the investigated pure compounds in this work characterise them to be in the semi-volatile organic compound (SVOC) category; hence they will be present both in the gaseous and condensed phase in the atmosphere. This implied that all these compounds will be susceptible for gas phase OH radical oxidation that was demonstrated to be an important process for the complex mixtures investigated in the smog chamber facilities. The OH chemistry was also influencing the volatility of aerosol produced in G-FROST by ozonlysis. Regarding photooxodation of amines the aerosols produced under high NOx conditions initially mimicked the pure salts but was efficiently transformed by oxidation into an aerosol with similar volatility properties as observed in the terpene oxidation experiments.Item Experimental studies of cluster ions containing water, ammonia, pyridine and bisulphate(2011-10-06) Ryding, MauritzMolecular cluster ions are fascinating subjects of study. Bridging the size gap between molecules and bulk, they often display non-trivial size dependent behaviour and properties. As an example, for some cluster types there are certain sizes that are found in unusually high abundance in a produced cluster distribution, these are referred to as gmagic numbers h. Apart from being interesting in their own right and serving as useful model systems in a number of applications, molecular clusters have a very real and important role in the vast and dynamic system we refer to as the atmosphere. Molecular clusters act as precursors for the formation of atmospheric particles. As such, it is necessary to learn as much as possible about the formation, growth, physical properties and chemistry of these clusters, because the particles they form will ultimately have a large effect on the global climate. This work investigates the properties of some ionic molecular clusters and their gas phase reactions with heavy water and ammonia, and also the effects of collision induced dissociation on air. This is done in cluster beam experiments, using two different experimental setups. The first instrument is a quadrupole-time-of-flight instrument, consisting of an electrospray ion source, a quadrupole mass filter, a collision cell and a time-of-flight mass spectrometer. In this instrument, relative reaction cross sections were measured for H+(H2O)n, H+(NH3)1(H2O)n and H+(pyridine)1.3(H2O)n colliding with D2O; and for H+(H2O)n, H+(pyridine)1.2(H2O)n and H+(NH3)1(pyridine)1(H2O)n colliding with NH3. The results for the reaction H+(pyridine)1(H2O)n + NH3 were used to improve a kinetic model of the atmospheric positive ion composition. Abundance spectra and evaporation patterns were recorded for all clusters. It was found that protonated clusters containing water and pyridine do not have magic numbers in the investigated size range (. 1500 u), unlike clusters consisting of water, pyridine and ammonia. Furthermore the magic numbers of H+(NH3)1(pyridine)1(H2O)n were the same as those recorded for H+(NH3)1(H2O)n. Cluster reactions with D2O proceed through a short-lived reaction complex. The clusters add the heavy water molecule and subsequently lose a D2O, HDO or H2O molecule; the latter two reaction channels are associated with a cluster mass increase of one or two atomic mass units, respectively. The formation of a HDO species in a cluster requires proton mobility, and is known to occur in H+(H2O)n clusters. The reaction channel leading to formation of HDO was not observed for protonated water clusters containing an ammonia or pyridine molecule, which is attributed to the proton being bound in place by the Bronsted base. However, the experiments indicate proton mobility in clusters with two or three pyridine molecules, H+(pyridine)2.3(H2O)n. Quantum chemical calculations suggest that this may be due to transfer of the proton to a water molecule, forming H3O+, or due to proton transfer between the two pyridine molecules along a wire of hydrogen bonds. The second instrument is a double sector instrument, having a magnetic sector, a collision cell and an electrostatic sector. Collision induced dissociation of H+(NH3)m(H2O)n clusters (m = 4.6) indicate that clusters having six NH3 prefer to lose NH3, while clusters with four or five NH3 prefer to lose H2O.Item Synthesis of Novel Inhibitors of IdeS, a Bacterial Cysteine Protease Including Studies of Stereoselective Reductive Aminations(2011-09-30) Berggren, KristinaAbstract The cysteine protease IdeS is an IgG degrading enzyme secreted by the bacterium Streptococcus pyogenes to evade the human immune system. In this thesis several inhibitors of IdeS have been synthesized and evaluated. Such inhibitors should be highly useful when elucidating the detailed mechanism of IdeS action. They might also have a potential as treatment of acute and severe infections caused by the bacteria. Further, IdeS has a therapeutic application of its own due to the proteolytic ability and an IdeS inhibitor might contribute during the development. Only irreversible, unselective inhibitors of IdeS were known five years ago. In this thesis, three strategies with the aim to synthesize and identify more inhibitors have been undertaken. Focus was first set on compounds with a substructure resembling the known inhibitors but with reversible warheads, i.e. nitrile, azide and aldehyde functions. The aldehyde derivatives were found to provide the first reversible inhibitors of IdeS. Then, to avoid covalent interactions and obtain more selective inhibitors, a substrate based strategy was undertaken. A 3-aminopiperidine fragment was used as replacement of either of the two residues adjacent to the scissile bond in IgG. Such fragments can be synthesized from N-protected 3-aminopiperidone and amino acid esters in reductive aminations in which a stereogenic center is formed. A series of di-, tri- and tetrapeptide analogues, together with eight peptides covering the cleavage site of IgG, were screened for their capacity to inhibit the cysteine proteases IdeS, SpeB and papain. Several analogues showed inhibition capacity, two compounds showed also high selectivity for IdeS. In contrast, none of the tested peptides showed any inhibition. Computational docking studies indicate that the identified IdeS peptide analogues and the non-active peptides do not share the same binding site in IdeS. Probably, the piperidine moiety hinders the inhibitor to enter the catalytic site. A more detailed study of the stereoselectivity in the reductive aminations affording the 3-aminopiperidine fragment showed that a large protecting group (trityl) together with a large reducing agent (NaBH(O-2- ethylhexanoyl)3) gave the highest diastereomeric ratio. The highest ratio obtained was 21:79 when Lproline methyl ester was used. The newly formed stereogenic center had the R-configuration, determined by chemical correlation. Computer based conformational analysis combined with Boltzmann distribution calculations implies an axial attack by the reducing reagent on the intermediary imine. To improve the potency of the two identified di- and tripeptide analogues synthetic routes to conformationally restricted N-containing bicyclic derivatives was undertaken in a third strategy. Five compounds with different bicyclic scaffolds were screened for their inhibition capacity towards IdeS and papain. One of the compounds was able to inhibit the first step of proteolytic cleavage of IgG by IdeS, a process usually completed in seconds.Item Chemical and Dermatological Aspects of UV-absorbing Compounds(2011-09-27) Karlsson, IsabellaABSTRACT The sun’s UV radiation is necessary for the existence of life on earth. However, too much UV exposure can lead to the development of skin cancer. Therefore, sunscreens are often used by the general population as protection from excessive UV radiation. Unfortunately, many of the chemical UV-filters that are used in sunscreens today have the ability to induce contact and photocontact allergy. In this work two different chemical UV-filters together with the anti-inflammatory drug ketoprofen, all known to induce allergic reactions, have been studied to better understand the reason for these adverse effects. In addition, a synthetic route to the natural UV-filter scytonemin has been developed. One of the most commonly used UVA-filters today is the well known photoallergen 4-tert-butyl-4’-methoxy dibenzoylmethane. We showed that it degrades when irradiated with UV light and that several different photodegradation products are formed. Of particular interest were arylglyoxals and benzils because they were unexplored as potential contact allergens. The benzils were found to be cytotoxic rather than allergenic, whereas the arylglyoxals were found to be strong sensitizers in the murine local lymph node assay (LLNA) used to assess their allergenic potency. Photocontact allergy to dibenzoylmethanes is therefore probably caused by the arylglyoxals that are formed upon photodegradation. Chemical reactivity experiments showed that the arylglyoxals have the ability to form immunogenic complexes via an electrophilic-nucleophilic reaction with the amino acid arginine. A relatively new UV-filter on the market is octocrylene that has grown in popularity, due to its ability to stabilize other UV-filters such as 4-tert-butyl-4’-methoxy dibenzoylmethane. However, recent clinical reports suggest that it is the UV-filter that causes most allergic reactions. Patch and photopatch testing of 172 patients with suspected skin reactions to sunscreens or ketoprofen was performed and 23 of these patients displayed a positive test reaction to octocrylene. Five patients were diagnosed with contact allergy and 18 with photocontact allergy. Notably, many of these patients also displayed a photoinduced reaction to ketoprofen. Without UV radiation, octocrylene was classified as a moderate allergen in the murine LLNA and it was shown to reacted with amines like lysine via a retro-aldol condensation. In presence of UV radiation, octocrylene also reacted with amines but via acyl substitution resulting in a different product outcome than the reaction in the dark. Both the clinical studies and the chemical reactivity experiments thereby indicate that octocrylene has the ability to induce both contact and photocontact allergy. The apparent photocross-reactivity between octocrylene and ketoprofen observed in the clinical study could not be explained by the previous reactivity studies of octocrylene. Furthermore, according to other clinical reports, photosensitization to ketoprofen also leads to photocontact allergy to many other compounds. Ketoprofen was therefore irradiated in presence of five amino acid analogs and interestingly a reaction between the tryptophan and lysine analogs was substantially enhanced by ketoprofen. We believe that ketoprofen generates singlet molecular oxygen which activates the tryptophan analog that subsequently reacts with the lysine analog. The formation of an immunogenic complex not containing the allergen itself can explain many of the observed photocross allergies between ketoprofen and other structurally different compounds. In theory all compounds that are able to generate singlet molecular oxygen can promote the formation of the same immunogenic complex. Finally, the first total synthesis of the dimeric alkaloid scytonemin was developed. This natural occurring UV-filter enables the survival of different species of cyanobacteria in areas of intense solar radiation. The planed structure activity studies of scytonemin and derivatives thereof will hopefully lead to the development of a stable UV-filter that does not cause contact or photocontact allergy.Item On the Electrolyte Induced Aggregation of Concentrated Silica Dispersions - An Experimental Investigation Using the Electrospray Technique(2011-09-08) Johnsson, Ann-CathrinGels are weak, solid-like structures that arise when colloidal particles aggregate to form a network of particle clusters. A variety of colloidal systems that are important scientifically as well as in industrial applications are capable of gel formation, e.g. globular protein solutions, colloid-polymer mixtures, and metal oxides. Yet, the mechanisms of the gelation process are far from understood, and the investigation of the aggregation and gelation of colloidal dispersions is, therefore, of great importance. Especially, the size distribution and structure of the aggregates are known to affect the gelation, and the main focus of this thesis is to improve our understanding of the initial aggregate formation in concentrated silica dispersions. The electrospray-scanning mobility particle sizer (ES-SMPS) technique has previously been demonstrated to be a valuable method for size distribution analysis of pure colloidal dispersions. Here, the ES-SMPS method was used to monitor the size distribution variation during electrolyte induced slow aggregation of concentrated silica dispersions. Number size distributions provide information about the primary particles as well as the formed aggregates. The influence of the ion specificity, as well as three initial particle morphologies, on the aggregation behaviour was investigated. Moreover, the aggregate diameters obtained by the ES-SMPS method were compared to the those obtained by other techniques such as scanning electron microscopy (SEM) and in situ small angle X-ray scattering (SAXS). The initial aggregate formation could be monitored accurately using the ES-SMPS method and compact, nearly spherical aggregates were observed for two of the initial morphologies. It was concluded that these resulted from a dynamic aggregation process where the aggregates broke and reformed several times prior to the gelation. More elongated aggregates were observed in the third dispersion; these aggregates were more rapidly stabilized by interparticle bonds and formed the most stable gel structures. The surface properties of the particles were found to affect the aggregate structure. Clear ion specific effects were observed; the most stable aggregates were formed in the presence of the least hydrated alkali ions, whereas the rate of gel stability increase was faster in the presence of the more strongly hydrated ions. As expected, the alkali ions adsorbed according to the direct Hofmeister sequence. A gel layer on the silica particle surfaces was identified for all dispersions investigated. The thickness of these layers were estimated using different techniques and found to be 2-4 nm thick depending on the dispersion.Item Analysis of phospholipids in cellular membranes with LC and imaging mass spectrometry(2011-05-20) Lanekoff, IngelaImaging mass spectrometry enables the creation of molecule specific images from the surface of a solid sample in vacuum. To solve the issue of bringing single cells into vacuum without altering their native distribution of molecules, a freeze fracture device that fits the time of flight secondary ion mass spectrometry (TOF-SIMS) IV instrument has been developed. This makes it possible to get a snapshot of the chemical distribution across frozen hydrated single cells that are only 10-20 µm in diameter. The cells of interest in this thesis are rat pheochromocytoma (PC12) cells. PC12 cells resemble and act like neurons in the sense that upon stimulation they release dopamine, which is a substance used for communication between neurons. In previous studies using these model cells, the rate of this release has been shown to change after the cells have been incubated with different phospholipids. To investigate the amount of phospholipids that have accumulated in the plasma membrane of PC12 cells after an overnight incubation, the combination of the freeze fracture device and the TOF-SIMS IV instrument was utilized. Relative to the endogenous phospholipid the results show that 0.5% of phosphatidylcholine (PC) and 1.3% of phosphatidylethanolamine (PE) had accumulated in the plasma membrane. Together with previous results on changes in the release of dopamine in PC12 cells, this suggests that the phospholipid composition of the plasma membrane of neurons is highly regulated. This gives a hint as to the importance of phospholipids during this highly important cellular process. The technique of liquid chromatography (LC) mass spectrometry (MS) does not provide molecular information in images but has the ability to separate similar molecules in a sample. This is of high importance when analyzing a specific molecule in a complex sample. Anaerobic ammonium oxidizing (anammox) bacteria reside in sediment on the ocean floor. These bacteria are highly important to the environment because they convert biologically available nitrogen into dinitrogen gas (N2), which is returned to the atmosphere. By denitrifying biologically available nitrogen they limit the risk of over fertilization in the ocean. They are also believed to contribute greatly to the global N2 production. By combining LCMS with an extensive sample clean up procedure a phospholipid biomarker for viable anammox bacteria has been used to detect the location of anammox bacteria in a sediment core sample.Item Colloidal interactions obtained from total internal reflection microscopy measurements and scattering data(2011-05-18) Nayeri, MohebThe scattering of radiation can be used to extract information about the interactions between colloidal (10^{-7}-10^{-3} cm radius) particles suspended in liquids. As colloidal interactions incorporate entropic effects they are weak and while system specific they are governed by a number of general mechanisms. Colloidal interactions can be studied to some extent by direct measurements or more indirectly by inferring information from measurements of some property of the system. In this thesis the principal experimental technique has been total internal reflection microscopy (TIRM), which is a very sensitive scattering technique. It allows for measurements of interaction energies between a single colloidal sphere and a flat surface in the area of 10^{-21} Joules. TIRM has been applied to show that high concentrations of non-ionic surfactant, often used at low concentrations to sterically stabilize colloidal particles, can cause particles to become physically attached by some bridging structure between the surface and particle. Another common stabilization mechanism widely used in colloidal systems is charge stabilization, whereby dissociated surface charges result in repulsion between particles and surfaces at low concentrations of electrolyte. Using TIRM a wide range of electrolytes and ionic strengths have been investigated, showing that the range of repulsion is given by the so-called Debye length for almost all situations that can be studied by TIRM. The exception is shown to be higher concentrations of 2:2 electrolytes, like MgSO4 and ZnSO4, in which repulsions are longer-ranged than expected. At high electrolyte concentrations attractive van der Waals interactions become important. When the interaction involves surfaces or particles of two different materials with a solvent with properties in-between those of the two materials, it is possible that the van der Waals interaction can become repulsive. Some support for this occurring in polar solvent mixtures under special conditions has been obtained by TIRM. Small colloidal particles can be used to induce effective interactions between larger particles and surfaces. A widely studied mechanism is depletion, which results from the imbalance in osmotic pressure when two surfaces come close enough together to exclude the small "depletant" spheres from the gap in between. TIRM was used to study the effect of concentration of charged depletant spheres and electrolyte on the depletion-like structural interactions between a large colloidal sphere and a flat surface. At high depletant concentrations an attraction is observed followed by a repulsive barrier as a function of separation distance, which is modeled using integral equation theory. Integral equation theory has also been used in modeling the interactions between oil-swollen surfactant micelles, so-called microemulsion droplets, based on non-ionic surfactant in water. Small-angle X-ray scattering data for a range of droplet concentrations were shown to be well described by a model based on an effective hard-sphere interaction, i.e. a short-ranged highly repulsive interaction, which is an example of an indirect method of obtaining information on colloidal interactions.Item Integrated biomechanical, electronic and topographic characterization of titanium dental implants(2011-05-11) Löberg, JohannaTitanium dental implants are medical devices used to restore the function entailed with the loss of one or several teeth. To obtain successful function over long periods, the dental implants must be sufficiently anchored in the bone to withstand the forces induced by for example chewing. Two important factors for obtaining high anchorage strength are i) the chemical composition of the material and ii) the implant design at all length scales. Topographical features on different length scales induce for example nucleation sites for collagen and minerals, cell attachment and biomechanical stimulation necessary to prevent bone resorption and eventually to gain bone. The design of nucleation sites at the titanium surface to stimulate bone growth is at the heart of the project presented in this thesis. The aim of the work is to in detail characterize the implant surface and design chemically as well as topographically modified surfaces by complementary experimental studies including electrochemical characterization and biomechanical models. The present thesis is based on three main topics of importance for dental implants: i) Surface topography measured with scanning electron microscopy and atomic force microscopy, ii) Biomechanical modelling and iii) Electronic properties of the surface oxide film investigated using impedance spectroscopy and cyclic voltammetry. Theoretical finite element studies have shown that the micro-topography of a surface can be designed to induce optimal biomechanical stimulation for bone formation. However, currently used topographical characterization methods for describing dental implant surfaces are insufficient to characterize the topography in the required detail to design such surfaces. In the present thesis, a method to investigate and describe the micro- to nanosized surface topographies is presented. In this method, complementary analysis techniques are used in combination with overlapping analysis areas and data filtering in order to obtain information from surface features in a wide range of length scales. Theoretical models have also been developed with the aim of evaluating the ability of micro- and nanoscaled surface features to induce retention strength with bone. By combining the characterization method and the theoretical models, an integrated characterization method is presented which can be used to design biomechanically optimized implant surfaces with suitable surface topography. Various modification techniques are used to alter the surface topography and as a secondary effect, the electronic properties of the oxide film will be altered. The effects on the biological response induced by changes in topography and electronic properties separately are therefore difficult to distinguish. The present thesis includes a study where the electronic properties of the surface oxide film were deliberately changed without significant changes in surface topography. The results show that the electronic properties of the oxide film have larger effects on the cellular attachment and apatite nucleation than a small change in topography and that a less insulating oxide film is preferable for titanium dental implants. The knowledge obtained from the biomechanical models and the electronic investigation was used to design well-characterized nanostructured surfaces created by coating titanium discs with titanium dioxide nanoparticles of different morphology and size. The bioactivity of the coated samples was evaluated by apatite formation and the results show that the coated samples induce earlier apatite nucleation and form thicker apatite layers than the uncoated reference. The results obtained and presented in this thesis suggest that dental implants should have roughness at different length scales in combination with formation of a thin defect rich oxide. Such an implant is still to be designed and tested under realistic conditions.Item Regulation of electron donation to photosystem 1(2011-05-09) Farkas, DanielPhotosynthesis is the process where energy from light is used to catalyze the formation of energy-rich molecules such as NADPH and ATP. These reactions are in fact the result of a long series of electron-transfer reactions where water molecules are the source of electrons and NADP+ is the terminal electron acceptor. A consequence of the electron-transfer reactions is the translocation of protons across a phospholipid membrane. This proton gradient in turn drives ATP synthase to catalyze the formation of ATP from ADP. From this chain of events, molecular oxygen is a byproduct, leading to the oxygen-rich atmosphere we live in today. In this thesis, the objective has been to study the specific protein-protein interaction between the electron-donor protein plastocyanin (Pc) and the photosystem 1 (PSI) subunit PsaF. The physiological regulation of this interaction has been a major subject and hence should be kept in mind to understand the purpose of the different papers and studies reported here. In paper I we report on the cloning, expression and characterization of the luminal domain of spinach PsaF. Characterization by several different biophysical techniques revealed a protein domain which is very dynamic and consistent with molten-globule like structural features. A disulphide bridge formed between cysteine residues 8 and 63 appeared to have a major role in stabilizing the tertiary fold of this domain. Site-directed mutagenesis and zero-length cross-linking revealed a native-like interaction with Pc, strongly dependent on the electrostatic character of the two proteins. The findings in paper I led us to investigate whether light-induced changes in the Mg(II) content in the chloroplast lumen can modulate the electron donation to PSI, in particular the electrostatic interaction between Pc and PsaF. Using NMR and EPR spectroscopy to characterize the Pc-PsaF complex and the one formed between Pc and Mg(II) we could observe similar binding constants. A competitive effect could be observed for the binding of Mg(II) to the Pc-PsaF complex, hence suggesting that the two interact with the same region of Pc. By studying the paramagnetic relaxation enhancement of the Mg(II) analogue, Mn(II), and its effect on Pc’s 15N-HSQC spectra, we could calculate the structure of the transient Pc-Mn(II) complex. The results presented suggest a specific binding site for Mg(II) that may regulate the binding of Pc to PSI in vivo. In paper III, we show that the luminal domain of PsaF is a target for thioredoxin-mediated reduction of the disulphide bridge formed between cysteines 8 and 63. Furthermore, we show that the thiolated form of PsaF has a lower affinity towards reduced Pc than when the disulfide bridge is intact. Time-resolved absorbance measurements and fluorescence electrophoresis show that oxidized Pc can re-oxidize PsaF and thus restore the active form of this domain. The PSI subunit PsaN is a weak modulator of the electron donation from Pc to PSI. In paper IV we present a methodology for the recombinant production of this protein subunit. Problems with unspecific proteolysis and degradation by Escherichia coli proteases are tackled.Item AQUAPORINS: Production Optimization and Characterization(2011-05-05) Öberg, FredrikAquaporins are water facilitating proteins embedded in the cellular membranes. Such channels have been identified in almost every living organism – including humans. They are vital molecules and their malfunction can lead to several severe disorders. An increased understanding of their structure, function and regulation is of utmost importance for developing current and future drugs. The first problem to overcome is to acquire the proteins in sufficient amounts to enable characterization. To achieve this, proteins are often produced in a host organism. One of the most successful hosts for recombinant overproduction is the yeast Pichia pastoris. Using this yeast we could obtain exceptional yield of aquaporin 1, whereas some others were below the threshold needed for successful subsequent characterization. In this process, we have established methods allowing fast and accurate determination of the initial production yield. Furthermore, we optimized the yield for low producing targets, enabling studies of proteins previously out of reach, exemplified with human aquaporin 4. Characterization has been performed on aquaporins obtained in sufficient quantities, and the functionality of aquaporin 1, 5 and 10 has been assessed. Furthermore, a glycosylation was found to stabilize the aquaporin 10 tetramer although only a minority of the monomers where modified. Moreover, we used protein crystallography to determine the three dimensional structure of a hAQP5 mutant, providing insight into regulation of the protein by trafficking. Taken together, these results provide insight into factors directing high production of eukaryotic membrane proteins. The subsequent characterization, including functional and structural determination, reveals new knowledge about aquaporin activity and regulation.Item Absolute Asymmetric Synthesis(2011-04-18) Lennartson, AndersAbsolute asymmetric synthesis is the synthesis of optically active products from achiral or racemic precursors only. This has generally been regarded as impossible and is relevant in the discussion of the origins of biomolecular homochirality. A possible route to absolute asymmetric synthesis involves total spontaneous resolution, which is possible for stereochemically labile substances which crystallise as conglomerates (i.e. the enantiomers crystallise in separate crystals). Using total spontaneous resolution it was, for the first time, possible to prepare bulk-quantities of configurationally labile five-, seven-, and nine-coordinate enantiomers, containing only achiral ligands. Previously, only four- and six-coordinate complexes have been prepared enantiomerically pure in bulk quantities. Spontaneous resolution of eight-coordinate complexes has also been reported. It was also possible to perform total spontaneous resolution of a diaryl sulphide, an octanuclear organo(oxo)zinc complex, and a diindenylzinc complex. In the case of a helical coordination polymer based on copper(I) chloride and triallylamine, it was found that repeated synthesis always yielded an excess of the same enantiomer, possibly due to the influence of cryptochirality. It has previously been practically impossible to measure enantiomeric excesses in stereochemically labile microcrystalline samples. A method utilising quantitative solid-state CD spectroscopy has been introduced to solve this problem. In the case of the chiral organometallic reagent di(3-picoline)di(1-indenyl)zinc, it was possible to perform reactions with N-chlorosuccinimide in the presence of methanol and p-benzoquinone yielding optically active stereochemically inert 1-chloroindene in high yield and high enantiomeric excess (up to 89% ee). During the cause of theses studies, three cases of concomitant crystallisation of racemic and chiral phases have been discovered. This is a rare phenomenon of considerable interest e.g. in structure prediction. The first synthetic route to well-defined hydridoalkylzincates is also reported.Item Homochiral Crystals for Selective Synthesis(2011-04-13) Kokoli, TheonitsaSix new coordination compounds were prepared from monodentate sulfide ligands and copper(I) halides and their crystal structures were determined by single-crystal X-ray diffraction. The aim was to prepare coordination polymers that crystallize as conglomerates in order to use them in total spontaneous resolution. The anionic and neutral ligands were varied in order to examine how they would affect the crystallization. All six complexes formed racemic crystals, and five of them were polymeric. Bidentate sulfide ligands were used to increase the possibility of obtaining a coordination polymer. Five new complexes were prepared and structurally characterized by single crystal X-ray diffraction. Three of the complexes formed coordination polymers but none of them crystallized as a conglomerate. Tetrahedral metal complexes have been resolved by total spontaneous resolution for the first time. A cationic silver(I) complex with a bidentate sulfide ligand was prepared and it crystallized as a conglomerate. Enantioenriched crystal batches were obtained with enantiomeric excesses up to 90 %. Three chiral Ru(II) complexes with bidentate sulfide ligands were prepared and all three crystallized as conglomerates. They were used in absolute asymmetric synthesis and oxidized enantioselectively. The oxidations resulted in a selectivity of > 98% without the use of a chiral catalyst. One of the Ru(II) complexes isomerizes when exposed to light. Four new phases, containing one or both isomers, co-crystallized from the same solution.