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Abstract Coronary heart disease (CHD) is a clinical manifestation of atherosclerosis. It is a major cause of mortality and morbidity both in Finland and globally. Even after the best known treatments a significant residual risk of CHD remains. A low plasma HDL cholesterol level (HDL, high-density lipoprotein) is a common lipid abnormality in patients affected by premature CHD and also a component of the metabolic syndrome, a cluster of risk factors for atherosclerosis associated with central obesity. In this study, a phenotype of low HDL cholesterol level and premature CHD was investigated in two Northern Finnish family populations. The aim was to find new biological factors accounting for the elevated CHD risk in the phenotype. In the subjects of family population I, plasma levels of antibodies (IgG, IgM, IgA) against experimental epitopes (malondialdehyde-acetaldehyde-modified, copper-oxidized) of oxidized LDL (low-density lipoprotein) particles were measured. In the subjects of family population II, capacity of HDL fractions (total HDL, HDL2 and HDL3) to accept cholesterol from a THP-1 experimental foam cell model was assayed (cholesterol efflux). In addition, a phospholipid composition of their HDL fractions (HDL2 and HDL3) was measured using liquid chromatography-mass spectrometry. The antibody levels were not related to CHD or to HDL cholesterol level. Instead, the cholesterol efflux to HDL2 fraction was clearly impaired in CHD, which was associated with the low HDL cholesterol level of the patients. The impaired cholesterol efflux to HDL2 fraction was primarily in conjunction with the metabolic syndrome. The phospholipid composition of HDL fractions was different between the affected and the non-affected subjects. As an example, characteristic of the metabolic syndrome were elevated contents of palmitic, palmitoleic or oleic acids relative to linoleic acid in lysophosphatidylcholines and phosphatidylcholines. In conclusion, the HDL fraction is both functionally and compositionally modified in the phenotype of low HDL cholesterol level and premature CHD. Especially the cholesterol efflux capacity of the HDL2 fraction and thus its many functional properties may be impaired. There are many characteristic features in the phospholipid composition of the HDL in the phenotype which were detected in HDL2 and HDL3 fractions. / Tiivistelmä Sepelvaltimotauti on ateroskleroosin kliininen ilmenemismuoto. Se on merkittävimpiä kuolleisuuden ja sairastavuuden aiheuttajia niin Suomessa kuin maailmalla. Parhaillakin tunnetuilla hoidoilla sepelvaltimotaudille jää huomattava jäännösriski. Plasman matala HDL-kolesterolitaso (HDL, high-density lipoprotein) on yleinen lipidipoikkeavuus varhaista sepelvaltimotautia sairastavilla ja myös eräs metabolisen oireyhtymän, eli keskivartalolihavuuteen liittyvän ateroskleroosin riskitekijäkasauman, komponentti. Tässä väitöskirjassa tutkittiin matalan HDL-kolesterolitason ja varhaisen sepelvaltimotaudin fenotyyppiä kahdessa pohjoissuomalaisessa sukuaineistossa. Tavoitteena oli löytää uusia biologisia tekijöitä fenotyypin kohonneen sepelvatimotautiriskin taustalta. Ensimmäisen aineiston henkilöiden plasmasta mitattiin vasta-ainetasoja (IgG, IgM, IgA) LDL-hiukkasten (LDL, low-density lipoprotein) kokeellisia hapettuneita epitooppeja (malonidialdehydi-asetaldehydi-modioitu ja kuparilla hapetettu LDL) vastaan. Toisessa aineistossa mitattiin henkilöiden HDL-fraktioiden (kokonais-HDL, HDL2 ja HDL3) kykyä saada aikaan kolesterolin ulosvirtausta kokeellisesta THP-1 vaahtosolumallista. Lisäksi heidän HDL-fraktioidensa (HDL2, HDL3) fosfolipidikoostumus mitattiin nestekromatografi-massaspektrometri-laitteistolla. Vasta-ainetasot eivät liittyneet sepelvaltimotautiin tai HDL-kolesterolitasoon. Sen sijaan kolesterolin ulosvirtaus HDL2-fraktioon oli selkeästi alentunut sepelvaltimotaudissa, mikä liittyi potilaiden pieneen HDL-kolesterolipitoisuuteen. Alentunut ulosvirtaus HDL2-fraktioon liittyikin ensisijaisesti metaboliseen oireyhtymään. HDL-fraktioiden fosfolipidikoostumus erosi terveiden ja sairaiden välillä. Esimerkiksi metabolisessa oireryhtymässä tunnusomaista oli lysofosfatidyylikoliinien ja fosfatidyylikoliinien sisältämän palmitiinihapon, palmitoleiinihapon tai oleiinihapon suurentunut määrä suhteessa niiden sisältämän linoleenihapon määrään. Loppupäätelmä on, että matalan HDL-kolesterolitason ja varhaisen sepelvaltimotaudin fenotyypin HDL-fraktio on sekä toiminnaltaan että koostumukseltaan muuntunut. Erityisesti HDL2-fraktion kyky saada aikaan kolesterolin ulosvirtausta ja näin ollen sen monet toiminnalliset ominaisuudet voivat olla alentuneet. Fenotyypin HDL:n fosfolipidikoostumuksessa on monia tunnusomaisia piirteitä, joita havaittiin sekä HDL2- että HDL3-fraktiossa.

New Zealand’s 5.3 million strong dairy herd returns approximately 106 million litres of urine to pasture soils daily. The urea in that urine is rapidly hydrolysed to ammonium (NH₄⁺), which is then nitrified, with denitrification of nitrate (NO₃⁻) ensuing. Nitrous oxide (N₂O), a potent greenhouse gas (GHG), is produced via nitrification and denitrification, which are enzyme-catalysed processes mediated by soil microbes. Thus microbes are linked intrinsically to urine patch chemistry. However, few previous studies have investigated microbial dynamics in urine patches. Therefore the objective of these four experiments was to investigate the effects on soil microbial communities of cow urine deposition. Methods used included phospholipid fatty acid (PLFA) analyses of microbial community structure and microbial stress, dehydrogenase activity (DHA) assays measuring microbial activity, and headspace gas sampling of N₂O, ammonia (NH₃) and carbon dioxide (CO₂) fluxes. Experiment 1, a laboratory study, examined the influence of soil moisture and urinary salt content on the microbial community. Both urine application and high soil moisture increased microbial stress, as evidenced by significant changes in PLFA trans/cis and iso/anteiso ratios. Total PLFAs and DHA showed a short-term (< 1 week) stimulatory effect on microbes after urine application. Mean cumulative N₂O-N fluxes were 2.75% and 0.05% of the nitrogen (N) applied, from the wet (70% WFPS) and dry (35% WFPS) soils, respectively. Experiment 2, a field trial, investigated nutrient dynamics and microbial stress with plants present. Concentrations of the micronutrients, copper, iron and molybdenum, increased up to 20-fold after urine application, while soil phosphorus (P) concentrations decreased from 0.87 mg kg ⁻¹ to 0.48 mg kg⁻¹. Plant P was also lower in urine patches, but total PLFAs were higher, suggesting that microbes had utilised the available nutrients. Microbial stress again resulted from urine application but, in contrast to experiment 1, the fungal biomass recovered after its initial inhibition. Studies published during the course of this thesis reported that hippuric acid (HA) and its hydrolysis product benzoic acid (BA) significantly reduced N₂O-N emissions from synthetic cow urine, thus experiment 3 investigated this effect using real cow urine. Cumulative N₂O-N fluxes were 16.8, 5.9 and 4.7% of N applied for urine (U) alone, U+HA and U+BA, respectively. Since NH₃-N volatilisation remained unchanged, net gaseous N emissions were reduced. Trends in total PLFAs and microbial stress were comparable to experiment 1 results. Experiment 4 studied HA effects at different temperatures and found no inhibition of N₂O-N fluxes from HA-amended urine. However, mean cumulative N₂O-N fluxes were reduced from 7.6% of N applied at 15–20°C to 0.2% at 5–10°C. Total cumulative N emissions (N₂O-N + NH₃-N) were highest at 20°C (17.5% of N applied) and lowest at 10°C (9.8% of N applied). Microbial activity, measured as potential DHA, increased with increasing temperature. This work has clearly shown that the stimulation and inhibition of the soil microbial community by urine application are closely linked to soil chemistry and have significant impacts not only on soil nutrient dynamics but also on N₂O-N emissions and their possible mitigation.

<p>Specific and non-specific interactions of solutes with immobilized biomembranes were studied using chromatographic methods. Liposomes, proteoliposomes and red blood cell (RBC) membrane vesicles were immobilized by a freeze-thawing procedure, whereas whole RBCs were adsorbed in the gel beds using electrostatic interaction, binding to wheat germ agglutinin (WGA) or the streptavidin-biotin interaction. </p><p>Superporous agarose gel with coupled WGA was the most promising matrix for RBC adsorption and allowed frontal chromatographic analyses of the cells for about one week. Dissociation constants for the binding of cytochalasin B and glucose to the glucose transporter GLUT1 were determined under equilibrium conditions. The number of cytochalasin B-binding sites per GLUT1 monomer was calculated and compared to corresponding results measured on free and immobilized membrane vesicles and GLUT1 proteoliposomes. This allowed conclusions about the protein´s binding state <i>in vitro</i> and <i>in vivo</i>. </p><p>Partitioning of drugs into biomembranes was quantified and the system was suggested as a screening method to test for possible intestinal absorption of drug candidates. We also studied how membrane partitioning of drugs is affected by the presence of integral membrane proteins or of charged phospholipids.</p><p>An attempt to combine the theory for specific binding and membrane partitioning of solutes in a single equation is briefly presented. </p>

<p>The human glucose transporter GLUT1 is abundant in red blood cells, the blood-brain barrier and epithelial cells, where it mediates the transport of the energy metabolite, glucose. In the present work some properties of GLUT1, including affinity binding of both substrates and inhibitors, transport rates as well as permeabilities of aromatic amino acids and drug-membrane interactions were analyzed by chromatographic methods.</p><p>Reconstitution by size-exclusion chromatography on Superdex 75 from a detergent with a low CMC that provides monomeric GLUT1 was examined regarding D-glucose- and CB binding as well as D-glucose transport. Upon steric immobilization in Superdex 200 gel beads, residual detergent could be washed away and dissociation constants in the same range as reported for binding to GLUT1 reconstituted from other detergents were obtained. The transport rate into the GLUT1 proteoliposomes was low, probably due to residual detergent. Binding to GLUT1 at different pH was analyzed and the affinity of glucose and GLUT1 inhibitors was found to decrease with increasing pH (5–8.7). The average number of cytochalasin B-binding sites per GLUT1 monomers was, in most cases, approximately 0.4. GLUT1 may work as a functional monomer, dimer or oligomer. To determine whether GLUT1 was responsible for the transport of the aromatic amino acids tyrosine and tryptophan, uptake values and permeabilities of these amino acids into liposomes and GLUT1 proteoliposomes were compared to the permeabilities of D- and L- glucose in the same systems. Dihydrocytochalasin B was identified to be a new inhibitor of tyrosine and tryptophan transport into red blood cells. Ethanol turned out to inhibit the specific binding between CB and GLUT1 and also to decrease the partitioning of CB and drugs into lipid bilayers. A capacity factor for drug partitioning into membranes that allows comparison between columns with different amount of immobilized lipids was validated, and turned out to be independent of flow rate, amount of lipids and drug concentration in the ranges tested.</p>

The human glucose transporter GLUT1 is abundant in red blood cells, the blood-brain barrier and epithelial cells, where it mediates the transport of the energy metabolite, glucose. In the present work some properties of GLUT1, including affinity binding of both substrates and inhibitors, transport rates as well as permeabilities of aromatic amino acids and drug-membrane interactions were analyzed by chromatographic methods. Reconstitution by size-exclusion chromatography on Superdex 75 from a detergent with a low CMC that provides monomeric GLUT1 was examined regarding D-glucose- and CB binding as well as D-glucose transport. Upon steric immobilization in Superdex 200 gel beads, residual detergent could be washed away and dissociation constants in the same range as reported for binding to GLUT1 reconstituted from other detergents were obtained. The transport rate into the GLUT1 proteoliposomes was low, probably due to residual detergent. Binding to GLUT1 at different pH was analyzed and the affinity of glucose and GLUT1 inhibitors was found to decrease with increasing pH (5–8.7). The average number of cytochalasin B-binding sites per GLUT1 monomers was, in most cases, approximately 0.4. GLUT1 may work as a functional monomer, dimer or oligomer. To determine whether GLUT1 was responsible for the transport of the aromatic amino acids tyrosine and tryptophan, uptake values and permeabilities of these amino acids into liposomes and GLUT1 proteoliposomes were compared to the permeabilities of D- and L- glucose in the same systems. Dihydrocytochalasin B was identified to be a new inhibitor of tyrosine and tryptophan transport into red blood cells. Ethanol turned out to inhibit the specific binding between CB and GLUT1 and also to decrease the partitioning of CB and drugs into lipid bilayers. A capacity factor for drug partitioning into membranes that allows comparison between columns with different amount of immobilized lipids was validated, and turned out to be independent of flow rate, amount of lipids and drug concentration in the ranges tested.

Specific and non-specific interactions of solutes with immobilized biomembranes were studied using chromatographic methods. Liposomes, proteoliposomes and red blood cell (RBC) membrane vesicles were immobilized by a freeze-thawing procedure, whereas whole RBCs were adsorbed in the gel beds using electrostatic interaction, binding to wheat germ agglutinin (WGA) or the streptavidin-biotin interaction. Superporous agarose gel with coupled WGA was the most promising matrix for RBC adsorption and allowed frontal chromatographic analyses of the cells for about one week. Dissociation constants for the binding of cytochalasin B and glucose to the glucose transporter GLUT1 were determined under equilibrium conditions. The number of cytochalasin B-binding sites per GLUT1 monomer was calculated and compared to corresponding results measured on free and immobilized membrane vesicles and GLUT1 proteoliposomes. This allowed conclusions about the protein´s binding state in vitro and in vivo. Partitioning of drugs into biomembranes was quantified and the system was suggested as a screening method to test for possible intestinal absorption of drug candidates. We also studied how membrane partitioning of drugs is affected by the presence of integral membrane proteins or of charged phospholipids. An attempt to combine the theory for specific binding and membrane partitioning of solutes in a single equation is briefly presented.

Arthropod hemocyanin and phenoloxidase are members of a group of proteins called the Type-3 copper oxygen-binding proteins, both possessing a highly conserved oxygen-binding site containing two copper atoms each coordinated by three histidine residues (Decker and Tuczek, 2000). Despite similarities in their active site, these proteins have very different physiological functions. Phenoloxidase possesses both tyrosinase and o-diphenoloxidase activity, and is predominantly involved in reactions which protect insects from infection (Kopàcek et al., 1995). Hemocyanin is a large multi-subunit protein with a primary function as a respiratory protein, reversibly binding and transporting molecular O2 (Decker and Rimke, 1998; Decker and Tuczek, 2000). Recently, it has been demonstrated in vitro that arthropod hemocyanin possesses an inducible phenoloxidase activity when incubated with denaturants, detergents, phospholipids or proteolytic enzymes. This activity appears to be restricted to only a few subunit types, and it has been hypothesised that it may be accompanied by conformational change which opens the active site increasing access for larger phenolic substrates (Decker and Jaenicke, 2004; Decker et al., 2001; Decker and Tuczek, 2000). This possibly suggests a dual role of hemocyanin in arthropods. The presented thesis deals with two distinct aims. The first was to isolate and sequence a phenoloxidase gene from the insect Spodoptera littoralis (Egyptian Cottonleaf Worm). Despite efforts, progress was hindered by a number of experimental problems which are outlined within the relevant chapters. The second aim was to characterise the mode of SDS induced phenoloxidase activity in arthropod hemocyanin from the ancient chelicerates Limulus polyphemus (horseshoe crab) and Eurypelma californicum (tarantula) and the more modern chelicerate Pandinus imperator (scorpion), using a number of biophysical techniques. The results indicated that the SDS induced phenoloxidase activity is associated with localised tertiary and secondary conformational changes in hemocyanin, most likely in the vicinity of the dicopper centre, thus enhancing access for larger phenolic substrates. Experiments indicate that copper remains associated with the protein during these structural changes; however the nature of the association is unclear. SDS concentrations approximating the CMC appeared critical in causing the necessary structural changes required for a significant increase in the detectable phenoloxidase activity to be exhibited.

The main objective of this thesis work was to gain understanding of the principles of biolubrication, focusing on synergistic effects between biolubricants. To this end surface force and friction measurements were carried out by means of Atomic Force Microscopy, using hydrophilic and hydrophobic model surfaces in salt solutions of high ionic strength (≈ 150 mM) in presence of different biolubricants. There was also a need to gain information on the adsorbed layers formed by the biolubricants. This was achieved by using a range of methods such as Atomic Force Microscopy PeakForce imaging, Quartz Crystal Microbalance with Dissipation, Dynamic Light Scattering and X-Ray Reflectometry. By combining data from these techniques, detailed information about the adsorbed layers could be obtained.The biolubricants that were chosen for investigation were a phospholipid, hyaluronan, lubricin, and cartilage oligomeric matrix protein (COMP) that all exist in the synovial joint area. First the lubrication ability of these components alone was investigated, and then focus was turned to two pairs that are known or assumed to associate in the synovial area. Of the biolubricants that were investigated, it was only the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) that was found to be an efficient lubricant on its own. Deposited DPPC bilayers on silica surfaces were found to be able to provide very low friction coefficients (≈ 0.01) up to high pressures, ≈ 50 MPa. A higher load bearing capacity was found for DPPC in the liquid crystalline state compared to in the gel state.The first synergy pair that was explored was DPPC and hyaluronan, that is known to associate on the cartilage surface, and we also noticed association between hyaluronan and DPPC vesicles as well as with adsorbed DPPC bilayers. By combining these two components a lubrication performance similar to that of DPPC alone could be achieved, even though the friction coefficient in presence of hyaluronan was found to be slightly higher. The synergy here is thus not in form of an increased performance, but rather that the presence of hyaluronan allows a large amount of the phospholipid lubricant to accumulate where it is needed, i.e. on the sliding surfaces.The other synergy pair was lubricin and COMP that recently has been shown to be co-localized on the cartilage surface, and thus suggested to associate with each other. Lubricin, as a single component, provided poor lubrication of PMMA surfaces, which we utilized as model hydrophobic surfaces. However, if COMP first was allowed to coat the surface, and then lubricin was added a low friction coefficient (≈ 0.03) was found. In this case the synergy arises from COMP facilitating strong anchoring of lubricin to the surface in conformations that provide good lubrication performance. / Huvudsyftet med det här avhandlingsarbetet var att öka förståelsen för den låga friktion som finns i vissa biologiska system, med fokus på synergistiska effekter mellan de smörjande molekylerna. För detta ändamål studerades ytkrafter och friktion med hjälp av atomkraftsmikroskopi. Mätningarna utfördes med hydrofila och hydrofoba modellytor i lösningar med hög salthalt (≈ 150 mM) i närvaro av smörjande biomolekyler. Det var också nödvändigt att få information om de adsorberade skikten av biomolekyler. Det åstadkoms med hjälp av en rad tekniker så som AFM PeakForce avbildning, kvartskristallmikrovåg, dynamisk ljusspridning och röntgen reflektometri. Genom att kombinera data från dessa tekniker erhölls detaljerad information om de smörjande skikten.De smörjande biomolekyler som valdes ut för studierna var en fosfolipid, hyaluronan, lubricin, and cartilage oligomeric matrix protein (COMP) vilka alla finns i synovialledsområdet. Först undersöktes den smörjande förmågan hos dessa komponenter var för sig, och sedan fokuserade vi på två par av biomolekyler som man vet eller antar bildar associationsstrukturer i synovialleder. Av de enskilda biomolekyler som undersöktes var det endast fosfolipiden 1,2-dipalmitoyl-sn-glycero-3-fosfokoline (DPPC) som visade sig vara en effektivt smörjande molekyl. Deponerade biskikt av DPPC på silikaytor gav upphov till mycket låga friktionskoefficienter (≈ 0.01) upp till höga pålagda tryck, ≈ 50 MPa. DPPC bilager i flytande kristallin fas visade sig ha högre lastbärande förmåga än DPPC bilager i geltillstånd.Det första synergistiska par som undersöktes var DPPC och hyaluronan vilka man vet associerar på broskytan, och vi visade att hyaluronan associerar med såväl DPPC vesiklar som med DPPC bilager. Genom att kombinera dessa två komponenter uppmättes en smörjande förmåga som var jämförbar med den som DPPC ensam uppvisar. Även om friktionskoefficienten var något högre i närvaro av hyaluronan. Synergieffekten här består inte av en bättre smörjande förmåga, utan istället gör närvaron av hyaluronan att de smörjande fosfolipiderna kan ansamlas i stora mängder där de behövs, dvs. på de glidande ytorna.Det andra synergiparet var lubricin och COMP vilka nyligen har visats vara lokaliserade på samma platser på broskytan, vilket tyder på att de associerar med varandra. På egen hand var lubricins smörjande förmåga av PMMA, våra hydrofoba modellytor, dålig. Emellertid, om COMP först adsorberades på PMMA och sedan lubricin tillsattes uppmättes en låg friktionskoefficient (≈ 0.03). I det här fallet består synergin av att COMP möjliggör en stark inbindning till ytan av lubricin i konformationer som ger god smörjande förmåga. / <p>QC 20141202</p> / Stiftelsen för strategisk forskning - SSF