A proof-of-concept for a novel microarray used to study protein-ligand interaction in real-time using label-free detection is presented. Many of todays commercially available instruments lack the ability to immobilize membrane proteins. At the same time, the pharmaceutical industry develops drugs directed towards membrane-bound receptors. The need to study drug-target kinetics and to be able to screen for new medical substances is high. To study the biomolecular interactions in real-time, imaging surface plasmon resonance (iSPR) is used. A patterned sensor surface with hydrophobic barriers assisting in the piezodispensing of NeutrAvidin with complex-bound biotin-ssDNA is created. Histidine-tagged proteins are immobilized at the vesicle surface using divalent nitrilotriacetic acid. The concept of the vesicle immobilization, the protein-binding to vesicles and the protein-ligand interaction is initially studied using a Biacore instrument. The dissociation of the ligand IFNα2 from its receptor ifnar-2 (wt) are in accordance with the literature. In the imaging SPR experiments, it is found that the dissociation of IFNα2 from the ifnar-2 (wt) receptor is slower than expected, probably due to rebinding of the ligand. It is also found that imidazole is needed to avoid vesicle-vesicle interaction. The immobilization of proteins had to be done on-line i.e. when the vesicles were bound to the surface. Depending on the mixture of receptors at the vesicle surface the affinity for the ligand was changed. The results achieved were reproducible.

Syfte Studien syftade till att undersöka om långvarig ihållande submaximal aktivering av Transversus Abdominis (TrA) påverkar dess anticipatoriska aktivering vid snabba viljemässigt utförda armrörelser. Metod I stående position utförde nio fysiskt aktiva kvinnor fem st snabba bilaterala armlyft från 0º till 90º axelflexion före och efter cirka 10 minuters ihållande submaximal aktivet i TrA samt efter 5 minuters vila. Elektromyografisk aktivitet (EMG) registrerades via två intramuskulära trådelektroder, placerade i höger TrA och två ytelektroder placerade på höger Deltoideus anterior. Buktrycket tros tillsammans med TrA stabilisera ryggraden vilket registrerades genom att en tryckgivare placerades i magsäcken. Resultat TrA aktiverades före Deltoideus både före och efter den långvariga aktiveringen. Initieringen av EMG-signalen i höger TrA hade oförändrat förhållande till initieringen av EMG-signalen i Deltoideus före och efter ihållande aktivitet samt efter 5 minuters vila. EMG amplituden i TrA var oförändrad både i baslinjefasen (600 ms före till 250 ms före deltoideus aktivering) och i den anticipatoriska fasen (100 ms före till 50 ms efter Deltoideus aktivering). Även buktrycksvärdet förblev oförändrat såväl vid baslinjen som i den anticipatoriska fasen. Slutsats Denna studie visade att centrala nervsystemet påbörjar aktiveringen av de innersta bukmusklerna före initieringen av armrörelserna och att detta förhållande ej påverkas av 10 minuters ihållande submaximal aktivering av TrA. Det finns inga tillgängliga metoder för direkt mätning av TrA:s mekaniska effekt, men eftersom buktrycket förblev oförändrad är det rimligt att tro att TrA:s kontraktilitet inte försämrats av den submaximala aktiveringen. / Aim The aim of the study was to investigate whether prolonged sustained sub maximal activation of Transversu Abdominis (TrA) influences its anticipitatory activation associated with fast voluntary shoulder flexion. Method In a standing position nine physically active female subjects (mean age of 26 ± 3 y) performed five rapid bilateral shoulder flexion from 0° to 90° shoulder flexion, before and after approximately 10 minutes of sustained submaximal activity in TrA as well as after 5 minutes rest. Electromyographic activity (EMG) was recorded using two intramuscular fine-wire electrodes placed in the right TrA and two surface electrodes placed over the Deltoideus anterior. Intra-abdominal pressure (IAP) was recorded intra-gastrically. Results TrA was activated prior to Deltoideus, before as well as after the sustained activation. The onset of TrA muscle activation relative to the onset of Deltoideus activation was not significantly different between before, directly after, or 5 minutes after the end of the sustained activity. The root mean square of the TrA EMG was unchanged both before arm lifts (baseline) and within the anticipatory window (100 ms before until 50 ms after Deltoideus onset). The IAP-value was unaffected in the baseline as well as in the anticipatory phase. Conclusion This study shows that the central nervous system begins activating the TrA slightly before initiating arm movements and that this behaviour is unaffected by a 10 min. sustained submaximal activation of TrA. There are no methods available for direct measurement of the mechanical output from TrA activation, but since IAP was unaffected it appears reasonable to conclude that the contractility of TrA is not deteriorated by the submaximal activation of TrA.

Mechanisms involved in the clearance of chylomicrons and aspects of the interactions at the vascular endothelium were studied in the rat. The poly-anion heparin, known to release lipoprotein lipase (LPL) from the vascular endothelium, enhanced the clearance of chylomicrons. Five minutes after heparin injection, the clearance of chylomicron triglycerides and retinyl esters was markedly accelerated. The rapid initial clearance was followed by a slower clearance of heavily lipolyzed chylomicrons. In contrast, one hour after heparin the clearance of both triglycerides and retinyl esters was retarded. This decreased removal of chylomicrons coincided with a decrease in the heparin releasable LPL activity, indicating that the previous release to plasma by heparin had resulted in net loss of functional LPL in the tissues. The poly-cation protamine released hepatic lipase and some LPL from their binding sites to plasma. One hour after protamine, plasma triglyceride levels were increased, indicating that chylomicron removal was impeded. It has been speculated that protamine inactivates LPL in vivo, but this was not the case. Ten minutes after injection of protamine normal amounts of LPL could be released by heparin. Thus, the accumulation of plasma triglycerides was not due to a rapid inactivation of LPL by protamine. LPL has specificity for sn-1,3-ester bonds. To investigate if this specificity is important in vivo, a lipid emulsion containing medium-chain fatty acids (MCFA) in the sn-1,3-position and long-chain fatty acids (LCFA) in the sn-2-position was synthesized, as well as an emulsion containing MCFA-TG mixed with LCFA-TGs (MMM/LLL). In vitro experiments showed large differences in the hydrolysis of the emulsions, but in vivo there were only small differences in the metabolism. To further study if lipid emulsions are cleared by the same mechanisms as chylomicrons, an emulsion was made by the same formulation as Intralipid® with addition of 3H-triolein and ,4C-cholesteryl ester. As measured by the removal of cholesteryl esters, the emulsion was cleared at the same rate as was chylomicrons. The triglyceride label was, however, removed more slowly from the emulsion droplets than from chylomicrons. Together with the lower recirculation of labeled free fatty acids (FFA) in plasma, this suggests that there was less lipolysis of the emulsion. The current view that removal of lipid emulsions in vivo is mainly dependent on LPL-mediated hydrolysis might thus not be correct. To further analyze the metabolism of chylomicrons, a compartmental model was developed. In this process, the distribution volume for chylomicrons was shown to be larger than the blood volume, a model for the metabolism of FFA in the rat was validated, and the full tissue distribution of injected chylomicrons was determined. According to the model, about half of the triglyceride label was removed from the circulation together with the core label while for the emulsion this number was about 80 %. In fasted rats all labeled fatty acids appeared to mix with the plasma FFA pool, while in fed rats about one-fifth of the fatty acids did not mix with the FFA but was apparently channeled directly to tissue metabolism. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1995, härtill 5 uppsatser.</p> / digitalisering@umu.se

Many important biological processes occur near or in membranes. The role of membranes is not merely confined to compartmentalization, they also form the matrix for membrane associated proteins and are of functional importance. Membrane associated proteins on the other hand require specific membrane properties for proper function. The interactions between membranes and proteins are thus of paramount importance and are at the focus of this work. To draw valid conclusions about the nature of such interactions the membrane mimetics required in biophysical methods must faithfully mimic crucial properties of biological membranes. To this end, new types of small isotropic bicelles which mimic plant and bacterial membranes were characterized by their size and lipid dynamics using solution-state NMR. Small isotropic bicelles are specifically well suited for solution-state NMR studies since they maintain a bilayer while being sufficiently small to conduct interpretable experiments at the same time. Monogalactosyl diacylglycerol and digalactosyl diacylglycerol, which are highly abundant in thylakoid membranes, were successfully incorporated into bicelles. Also, it was possible to make bicelles containing a lipid mixture extracted from Escherichia coli cells. A fundamental physical property of lipids in bilayers is their phase behaviour and thus the dynamics that lipids undergo in a membrane. Here, the dynamics of 13C-1H bonds in lipids were studied by nuclear spin relaxation. From such studies it was found that the glycerol backbone of lipids in bicelles is rigid while the flexibility of the acyl chain increases towards its end. Bulky head groups are rigid, while smaller head groups are more dynamic than the glycerol backbone. Acyl chain modifications, like unsaturations or cyclopropane moities, that are typically found in E. coli lipids, locally increase the rigidity of the acyl chain. Membrane interactions of a putative membrane anchor of the glycosyltransferase WaaG, MIR-WaaG, were studied by fluorescence methods, circular dichroism and solution-state NMR. It was found that MIR-WaaG binds to vesicles that mimic the anionic charge of E. coli inner membranes and that α-helical structure is induced upon interaction. The NMR-structure of MIR-WaaG agrees well with the crystal structure and from paramagnetic relaxation enhancement studies it could be concluded that a central part of MIR-WaaG is immersed in the membrane mimetic. Based on these results a model of the membrane interaction of WaaG is proposed where MIR-WaaG anchors WaaG to the cytosolic leaflet of the E. coli inner membrane via electrostatic interactions. These are potentially enhanced by membrane interactions of Tyr residues at the membrane interface and of hydrophobic residues inside the membrane.

In this thesis the colonization factor antigen I (CFA/I) of the enterotoxicogenic Escherichia coli (ETEC) is characterized biophysically with an optical tweezers. ETEC is the causative agent of travellers’ diarrhoea. The CFA/I is an adherence organelle, often named pili, mediating attachment to the epithelia of the small intestine, essential for infection. An optical tweezers were used to unfold the pili showing a steady state force of about 8 pN during unfolding. Also, the force dependence on unfolding velocity was studied. At velocities above 1.75 μm/s, the force increased linearly with the logarithmic velocity, in line with results from UPEC associated E. coli. The resulting force of CFA/I differs significantly from previously studied type 1, P and S pili which are in the range 25-30 pN. This is supposed to reflect differences in the host environment, i.e. the difference between gastrointestinal and urinary tract. The general behaviour however, seem to be the same for the different pili, in accordance with the similar macromolecular structure. In addition, the steady state force of the F1C pili of Escherichia coli was estimated to 28pN.

<p>The Amyloid beta peptide (Ab) is related to Alzheimer’s disease and is suggested to be the molecular pathogenic species of the disease, probably through the neurotoxic effect of Ab oligomers. Here the results from biophysical studies of Ab and fragments thereof, are presented. Pulsed field gradient NMR diffusion experiments show that Ab exists mainly as an unfolded monomer. In addition, the hydrodynamic radius of Ab suggests that Ab has residual secondary structure propensities. CD experiments reveal that Ab has a high propensity to adopt a polyproline type II (PII) helix at low temperature. NMR diffusion measurements as well as the 3JHNH values show that increasing the temperature from 4 C induces a structure transition from PII propensity to a beta strand propensity around 15 C and to a random coil conformation at higher temperature. The small hydrodynamic radius at low temperature may be explained by the presence of a population of a hairpin conformation as was suggested by MD simulations. 15N relaxation and secondary chemical shifts suggest that Ab consists of 6 structural regions, two regions with high PII propensity are separated by a highly mobile region located in the N-terminal part of the peptide. In the C-terminal part two regions with a propensity to adopt b-strand are located, separated by a mobile region. The structural propensities of soluble monomeric Ab agree well with the structure of the peptide in fibril aggregates as well as in SDS micelles. Ab binds zinc specifically and with high affinity. This interaction was studied using heteronuclear correlation experiments. The metal ligands were determined to be three histidines, 6,13 and 14 and the N-terminus. The Ab peptide also binds b-cyclodextrin and the combined use of NMR diffusion experiments and induced chemical shifts show that Ab has at least two binding sites for b-cyclodextrin, and the dissociation constants of these binding sites were determined.</p>

<p>Isotropic bicelles are disc-shaped aggregates of lipids and detergents, and are suitable model systems for high-resolution NMR studies of membrane-interacting peptides. In this thesis the structures for the two peptides motilin and transportan were determined by homonuclear ¹H methods in the presence of bicelles, and the structure of the bovine prion protein peptide (bPrPp) was solved in the presence of DHPC micelles. All of these peptides were found to be largely a-helical when bound to the model membranes. In subsequent experiments both motilin and transportan were shown to reside on the surface of the bicelles, whereas bPrPp is more likely to have a transmembrane configuration. </p><p>NMR translational diffusion experiments revealed that the isotropic bicelles studied here are very large objects compared to what is regularly indicated by high-resolution NMR spectroscopy. Furthermore, these studies showed that all three peptides examined interact strongly with bicelles. Investigation of the NMR-relaxation of labeled sites in the peptides motilin and penetratin demonstrated that the overall rotational correlation times for these peptides do not reflect the bicellar size. Such decoupling of NMR relaxation from the dependence of overall size is also seen for the dynamics of the lipid molecules in the bicelles. It is therefore concluded that the overall size is not the sole determinant of the linewidths in NMR spectra, but that extensive motions within the bicelles also exert significant effects. </p><p>Another interesting observation is that the membrane-bound structures of the peptides motilin, transportan, penetratin and bPrPp are very similar, even though these peptides have very different biological functions. In contrast, considerably more variation is observed in the membrane-positioning and molecular dynamics of these peptides. Since the bicelles have been found to induce differences in membrane positioning and molecular dynamics compared to micelles, these model membranes are likely to be important in order to enhance our understanding of the biological function of membrane interacting peptides.</p>

<p>In natural oxygen-producing photosynthesis solar energy is stored as chemical energy, in carbohydrates, fats and amino acids, using water as electron source. The large transmembrane protein complex, PSII, is the key enzyme in the light-driven reactions. Water oxidation is accomplished by a triad in PSII in which the Mn-cluster plays an important role. In the artificial photosynthetic system, nature’s photosynthesis will be mimicked such that hydrogen, a sustainable energy source, can be produced from solar energy and water alone. Since water oxidiation requires the catalytic activity of a Mn-cluster in photosynthesis, different artificially constructed manganese complexes are investigated. </p><p>The dinuclear ([Mn₂(II,III)L(µ-OAc)₂]ClO₄), where L is the X-anion of 2-(<i>N,N</i>-Bis(2-methylpyridyl)aminomethyl)-6-(<i>N</i>-(3,5-ditert-butylbenzyl-2-hydroxy)-<i>N</i>-(pyridylmethyl)aminomethyl)-4-methylphenol, an unsymmetric ligand with two coordinating phenolate groups, has been studied. The two Mn-ions are linked via a mono-µ-oxo bridge and two acetate ligands. Q-band Electron Paramagnetic Resonance was conducted on the Unsymmetric Mn₂(II,III) Complex. Aquired results show that the complex has a 2600 Gauss broad signal (11 400-14 000 Gauss) with 14-17 lines at g~2 and hyperfines of 120 Gauss. This is consistent with previous X-band studies. Q-band spectra of the Unsymmetric Mn(II,III) display increased hyperfine resolution compared to Qband spectra of the symmetric complex, Mn₂(bpmp)(µ-OAC)₂. This is noticeable since Unsymmetric Mn2(II,III) and Mn₂ (bpmp)(µ-OAC)₂ partly overlap in low-frequency experiments (X-band EPR). </p><p>Further investigations are yet to be expected. Nevertheless, the conducted thesis study provides important knowledge in the futuristic goal of building an artificial super-complex.</p>

<p>Implants are commonly used for orthopaedic and dental applications. There is however a problem with implants; they have a tendency to get loose after 10-15 years of usage. Bone that is not used will get weaker; this can be concluded from studies of people being immobilised or in microgravity. When an implant is put into bone, the surrounding bone does not experience any deformation and it will resorb. This is called stress shielding. Finally the implant will get loose. To avoid this problem we want to give electrical stimulation to the bone surrounding the implant. Electricity has been used before to stimulate bone, and it has been shown that immobilised bone can almost be maintained by using electric stimulation.</p><p>Piezoelectricity is a property of certain materials that make them generate electricity when they are deformed. When an implant is coated with a piezoelectric material, electrical stimulation can be achieved for the surrounding bone that is stress shielded.</p><p>In this diploma work, a test-equipment is built to stimulate cells. Cells will be grown on a piezoelectric plate that is bent by the test-equipment. Thus, the cells will be stimulated by both mechanical stress and electric potential since the piezoelectric material generates electricity when it is deformed. Piezoelectric samples and culture wells suitable for bending applications are prepared and tested in the equipment.</p><p>Some initial cell growth experiments have been performed to see that the material is suitable for cell growth.</p>

Isotropic bicelles are disc-shaped aggregates of lipids and detergents, and are suitable model systems for high-resolution NMR studies of membrane-interacting peptides. In this thesis the structures for the two peptides motilin and transportan were determined by homonuclear 1H methods in the presence of bicelles, and the structure of the bovine prion protein peptide (bPrPp) was solved in the presence of DHPC micelles. All of these peptides were found to be largely a-helical when bound to the model membranes. In subsequent experiments both motilin and transportan were shown to reside on the surface of the bicelles, whereas bPrPp is more likely to have a transmembrane configuration. NMR translational diffusion experiments revealed that the isotropic bicelles studied here are very large objects compared to what is regularly indicated by high-resolution NMR spectroscopy. Furthermore, these studies showed that all three peptides examined interact strongly with bicelles. Investigation of the NMR-relaxation of labeled sites in the peptides motilin and penetratin demonstrated that the overall rotational correlation times for these peptides do not reflect the bicellar size. Such decoupling of NMR relaxation from the dependence of overall size is also seen for the dynamics of the lipid molecules in the bicelles. It is therefore concluded that the overall size is not the sole determinant of the linewidths in NMR spectra, but that extensive motions within the bicelles also exert significant effects. Another interesting observation is that the membrane-bound structures of the peptides motilin, transportan, penetratin and bPrPp are very similar, even though these peptides have very different biological functions. In contrast, considerably more variation is observed in the membrane-positioning and molecular dynamics of these peptides. Since the bicelles have been found to induce differences in membrane positioning and molecular dynamics compared to micelles, these model membranes are likely to be important in order to enhance our understanding of the biological function of membrane interacting peptides.