<p>Dynorphin A, dynorphin B and big dynorphin are endogenous opioid neuropeptides. They play an important role in a wide variety of physiological functions such as regulation of pain processing and memory acquisition. Such actions are generally mediated through the κ-receptors. Besides opioid receptor interactions, dynorphins have non-opioid physiological activities which result in excitotoxic effects in neuropathic pain, spinal cord and brain injury. In order to gain insight into the mechanisms of the non-opioid interactions of dynorphins with the cell, spectroscopic membrane-interaction studies were performed. We demonstrated that big dynorphin and dynorphin A, but not dynorphin B, penetrated into cells. All dynorphins interact with the membrane model systems with weak membrane-induced secondary structure. Big dynorphin and dynorphin A induce membrane perturbation, calcein leakage and cause permeability of the membrane to calcium in large unilamellar vesicles (LUV). But dynorphins do not translocate in the LUV membrane model system and there is a strong electrostatic contribution to the interaction of the peptides with the membrane bilayer.</p><p>In the second part of this thesis we investigated the amyloid β(1-40) peptide (Aβ). This peptide is related to Alzheimer’s disease and its soluble oligomeric aggregates are reported to contribute to the pathology of the disease. In order to provide better insight into the aggregation processes we examined the membrane interaction of Aβ in a model system. Gradual addition of small amounts of sodium dodecyl sulfate to an aqueous solution gives rise to a secondary structure conversion of Aβ peptide. The conversion can be described as a two state process, from random coil to β-sheet with formation of high molecular mass complexes between peptide and detergent, possibly mimicking the behavior of the peptide when aggregating at a cell membrane surface. At high detergent concentrations there is a transition from β-sheet to α-helix conformation.</p>

Dynorphin A, dynorphin B and big dynorphin are endogenous opioid neuropeptides. They play an important role in a wide variety of physiological functions such as regulation of pain processing and memory acquisition. Such actions are generally mediated through the κ-receptors. Besides opioid receptor interactions, dynorphins have non-opioid physiological activities which result in excitotoxic effects in neuropathic pain, spinal cord and brain injury. In order to gain insight into the mechanisms of the non-opioid interactions of dynorphins with the cell, spectroscopic membrane-interaction studies were performed. We demonstrated that big dynorphin and dynorphin A, but not dynorphin B, penetrated into cells. All dynorphins interact with the membrane model systems with weak membrane-induced secondary structure. Big dynorphin and dynorphin A induce membrane perturbation, calcein leakage and cause permeability of the membrane to calcium in large unilamellar vesicles (LUV). But dynorphins do not translocate in the LUV membrane model system and there is a strong electrostatic contribution to the interaction of the peptides with the membrane bilayer. In the second part of this thesis we investigated the amyloid β(1-40) peptide (Aβ). This peptide is related to Alzheimer’s disease and its soluble oligomeric aggregates are reported to contribute to the pathology of the disease. In order to provide better insight into the aggregation processes we examined the membrane interaction of Aβ in a model system. Gradual addition of small amounts of sodium dodecyl sulfate to an aqueous solution gives rise to a secondary structure conversion of Aβ peptide. The conversion can be described as a two state process, from random coil to β-sheet with formation of high molecular mass complexes between peptide and detergent, possibly mimicking the behavior of the peptide when aggregating at a cell membrane surface. At high detergent concentrations there is a transition from β-sheet to α-helix conformation.

Cellular biophysics deals with the physical aspects of cell biology. This thesis presents a number of studies where mathematical models and data analysis can increase our understanding of this field. During recent years development in experimental methods and mathematical modeling have driven the amount of data and complexity in our understanding of cellular biology to a new level. This development has made it possible to describe cellular systems quantitatively where only qualitative descriptions were previously possible. To deal with the complex data and models that arise in this kind of research a combination of tools from physics and cell biology has to be applied; this constitutes a field we call cellular biophysics. The aim of this doctoral thesis is to develop novel approaches in this field. I present eight studies where quantitative modeling and analysis are involved. The first two studies concern cells interacting with their surrounding environment in the kidney. These cells sense fluid flow and respond with calcium (Ca2+) signals. The interaction between fluid and cells in renal tubular epithelium can be described by biomechanical models. This thesis describes a mathematical model of flow sensing by cilia with focus on the flow frequency response and time delay between the mechanical stress and the Ca2+ signaling response. Intracellular Ca2+ is kept at a very low level compared to the extracellular environment, while several intracellular compartments have higher Ca2+ concentration than the cytoplasm. This makes Ca2+ an efficient messenger for intra­cellular signaling, the process whereby signals are transduced from an extracellular stimulus to an intracellular activity such as gene expression. An important type of Ca2+ signaling is oscillations in intracellular Ca2+ concentration which occur due to the concerted interplay between different transport mechanisms within a cell. A study in this thesis examines ways to explain these mechanisms in terms of a mathematical model. Another study in the thesis reports that erythropoietin can regulate the water permeability of astrocytes and that it alters the pattern of Ca2+ oscillations in astrocytes. In this thesis the analysis of this Ca2+ signaling is described. Simulations described in one of the studies show how different geometries can affect the fluorescence recovery and that geometrically constrained reactions can trap diffusing receptors in dendritic spines. When separate time scales are present in a fluorescence revovery after photobleaching (FRAP) experiment the reaction and diffusion components can be studied separately. Applying single particle tracking methods to the migration trajectories of natural killer cells shows that there is a correlation between the formation of conjugates and transient confinement zones (TCZs) in these trajectories in vitro. TCZs are also present in in vivo experiments where they show strong similarities with the in vitro situation. This approach is a novel concept in data analysis methods for tracking immune cells. / Cellens biologiska fysik behandlar de fysikaliska aspekterna av cellbiologi. Denna avhandling presenterar ett antal studier där matematiska modeller och dataanalys kan öka vår förståelse av detta område. Under senare år har utvecklingen av experimentella metoder och matematisk modellering drivit mängden data och komplexiteten i vår förståelse av cellbiologi till en ny nivå. Denna utveckling har gjort det möjligt att beskriva cellulära system kvantitativt där endast kvalitativa beskrivningar tidigare var möjliga. För att hantera de komplexa data och modeller som uppstår i denna typ av forskning krävs en kombination av verktyg från fysik och cellbiologi; detta utgör ett område vi kallar cellens biologiska fysik. Syftet med denna avhandling är att utveckla nya metoder inom detta område. Jag presenterar åtta studier där kvantitativ modellering och analys ingår. De första två studierna behandlar hur celler interagerar med sin omgivning i njurarna. Dessa celler känner av ett vätskeflöde och svarar med kalcium (Ca2+)-signaler. Samspelet mellan vätska och celler i tubulärt njurepitel kan beskrivas med biomekaniska modeller. Denna avhandling beskriver en matematisk modell för flödeskänslighet hos cilier med fokus på flödesfrekvenssvar och tidsfördröjningen mellan den mekaniska påverkan och Ca2+-signaleringssvaret. Intracellulärt Ca2+ hålls på en mycket låg nivå jämfört med den extracellulära miljön, samtidigt som flera intracellulära delar har högre Ca2+-koncentrationen än cytoplasman. Detta gör Ca2+ till en effektiv bärare för intracellulär signalering, den process där signaler överförs från ett extracellulärt stimuli till en intracellulär händelse, exempelvis genuttryck. En viktig typ av Ca2+-signalering är de oscillationer i intracellulär Ca2+-koncentration som uppstår på grund av det ordnade samspelet mellan olika transportmekanismer i en cell. En studie  i denna avhandling undersöker olika sätt att förklara dessa mekanismer i form av en matematisk modell. En annan studie i avhandlingen rapporterar att erytropoietin kan reglera vattenpermeabilitet av astrocyter och att det ändrar mönstret av Ca2+-oscillationer i astrocyter. I denna avhandling beskrivs analysen av denna Ca2+-signalering. Simuleringar som beskrivs i en av studierna visar hur olika geometrier kan påverka fluorescensåterhämtning och att geometriskt begränsade reaktioner kan fånga in receptorer in i dendrittaggar. När separata tidsskalor förekommer i ett fluorescence revovery after photobleaching (FRAP)-experiment kan reaktions- och diffusionskomponenter studeras separat. Tillämpande av single particle tracking-metoder på naturliga mördarceller visar att det finns ett samband mellan bildandet av konjugat och transient confinement zones (TCZs) i dessa trajektorier in vitro. TCZs förekommer också i in vivo-experiment där de visar stora likheter med in vitro-situationen. Denna strategi är ett nytt grepp inom dataanalys-metoder för att spåra immunceller. / QC 20100726

The molecular motors of muscle are of potential interest in nanotechnology. These motors consist of the protein, myosin II interacting with actin filaments. It would be of interest to control the interaction between actin and myosin, e.g. in order to steer their direction of motion. Because these proteins are electrically charged their motion in a cell filled with a solution could potentially be controlled by an electric field. Here I have addressed several problems associated with experiments of this type. A main problem was found to be excessive heating of the solution. Another complication was electroosmotic flow and chemical reactions on the cell surface. The electric field can also cause electrophoretic motion of the proteins, which in some cases is undesired. The most effective way to reduce the heating of the solution was to keep the ratio between the cross sectional area of the cell and its cooling surfaces as small as possible. External cooling of the cell and keeping the ionic concentration in the solution as low as possible also prevented overheating. The electroosmotic flow could be stopped with agarose plugs at the cell openings and the surface reactions can probably be avoided if trimethylchlorosilane (TMCS) coated glass rather than nitrocellulose film is used for myosin adsorption. If electrophoretic motion turns out to be a problem it can be reduced/stopped with an electroosmotic flow in the opposite direction. A further conclusion of this study is that actin filaments may be oriented by relatively small field strengths whereas it can be necessary to use electric field strength of 1 MV/m or more to orient myosin. At this extremely high field strength the heat production, in a cell with a rectangular cross section, would probably will be to high. However, if a cell with a circular and very low cross sectional area, i.e. a capillary, is used the heating can possibly be held under an acceptable limit. / Nya generationer av datorer, digitalkameror, mobiltelefoner och annan elektronisk/teknisk utrustning tenderar att bli mindre utrymmeskrävande i förhållande till sin kapacitet i jämförelse med föregående modeller. Detsamma gäller också nya diagnostiska verktyg inom sjukvård, miljöövervakning m.m. Ett behov av aktiva komponenter av mindre format finns alltså på olika håll och möjligheten att skapa komponenter med proteiner som utgångspunkt har börjat undersökas. I denna studie fokuseras på aktin och myosin som tillsammans utgör de viktigaste proteinkomponenterna i skelettmuskler hos t.ex. människor och däggdjur. Dessa proteiners främsta uppgift är således att skapa en rörelse. Hur aktin och myosin fungerar tillsammans kan undersökas i konstgjorda testsystem (in vitro motility assay; IVMA), där proteinerna studeras utanför sin naturliga miljö. Vid den IVMA-metod som ligger till grund för denna undersökning förflyttar sig aktinet mer eller mindre okontrollerat ovanpå myosinet som fästs till en glasyta. För att aktinets rörelse skall bli tekniskt intressant måste denna rörelse kunna kontrolleras med viss noggrannhet. Då dessa proteiner är elektrisk laddade finns möjlighet att påverka/styra dem med elektromagnetiska kraftfält. Huvudsyftet med detta arbete har varit att undersöka om aktinets hastighet och rörelseriktning är möjlig att kontrollera med elektriska fält och vilka komplikationer som kan uppstå. Vid IVMA-försöken är aktinet och myosinet omgivna av en vattenbaserad saltlösning som är nödvändig för dessa proteiners funktion. Eftersom saltvatten är elektriskt ledande, så kommer en elektrisk ström att gå igenom saltlösningen när det elektriska fältet kopplas på. Den elektriska strömmen genom saltlösningen leder i sin tur till att lösningen värms upp. Risk finns alltså att saltlösningens temperatur stiger så mycket så att proteinerna upphör att fungera. Ett annat resultat av elektriska fält genom vattenbaserade lösningar är s.k. elektroosmotiskt flöde. Fenomenet elektroosmos innebär att lösningen försätts i en rörelse som är proportionell mot det elektriska fältets storlek. Vid kraftiga elektriska fält är det alltså möjligt att aktinet sköljs med i det elektroosmotiska flödet. Ytterligare en komplikation som kan uppstå vid elektriska fält genom IVMA-cellen är reaktioner i den beläggning som täcker glasytan vilken utgör botten på cellen. I detta examensarbete har en stor del av tiden ägnats åt att eliminera/reducera ovanstående oönskade bieffekter vid användandet av elektriska fält för att styra proteiner.

<p>The molecular motors of muscle are of potential interest in nanotechnology. These motors consist of the protein, myosin II interacting with actin filaments. It would be of interest to control the interaction between actin and myosin, e.g. in order to steer their direction of motion. Because these proteins are electrically charged their motion in a cell filled with a solution could potentially be controlled by an electric field. Here I have addressed several problems associated with experiments of this type. A main problem was found to be excessive heating of the solution. Another complication was electroosmotic flow and chemical reactions on the cell surface. The electric field can also cause electrophoretic motion of the proteins, which in some cases is undesired. The most effective way to reduce the heating of the solution was to keep the ratio between the cross sectional area of the cell and its cooling surfaces as small as possible. External cooling of the cell and keeping the ionic concentration in the solution as low as possible also prevented overheating. The electroosmotic flow could be stopped with agarose plugs at the cell openings and the surface reactions can probably be avoided if trimethylchlorosilane (TMCS) coated glass rather than nitrocellulose film is used for myosin adsorption. If electrophoretic motion turns out to be a problem it can be reduced/stopped with an electroosmotic flow in the opposite direction. A further conclusion of this study is that actin filaments may be oriented by relatively small field strengths whereas it can be necessary to use electric field strength of 1 MV/m or more to orient myosin. At this extremely high field strength the heat production, in a cell with a rectangular cross section, would probably will be to high. However, if a cell with a circular and very low cross sectional area, i.e. a capillary, is used the heating can possibly be held under an acceptable limit.</p> / <p>Nya generationer av datorer, digitalkameror, mobiltelefoner och annan elektronisk/teknisk utrustning tenderar att bli mindre utrymmeskrävande i förhållande till sin kapacitet i jämförelse med föregående modeller. Detsamma gäller också nya diagnostiska verktyg inom sjukvård, miljöövervakning m.m. Ett behov av aktiva komponenter av mindre format finns alltså på olika håll och möjligheten att skapa komponenter med proteiner som utgångspunkt har börjat undersökas. I denna studie fokuseras på aktin och myosin som tillsammans utgör de viktigaste proteinkomponenterna i skelettmuskler hos t.ex. människor och däggdjur. Dessa proteiners främsta uppgift är således att skapa en rörelse. Hur aktin och myosin fungerar tillsammans kan undersökas i konstgjorda testsystem (in vitro motility assay; IVMA), där proteinerna studeras utanför sin naturliga miljö. Vid den IVMA-metod som ligger till grund för denna undersökning förflyttar sig aktinet mer eller mindre okontrollerat ovanpå myosinet som fästs till en glasyta. För att aktinets rörelse skall bli tekniskt intressant måste denna rörelse kunna kontrolleras med viss noggrannhet. Då dessa proteiner är elektrisk laddade finns möjlighet att påverka/styra dem med elektromagnetiska kraftfält. Huvudsyftet med detta arbete har varit att undersöka om aktinets hastighet och rörelseriktning är möjlig att kontrollera med elektriska fält och vilka komplikationer som kan uppstå. Vid IVMA-försöken är aktinet och myosinet omgivna av en vattenbaserad saltlösning som är nödvändig för dessa proteiners funktion. Eftersom saltvatten är elektriskt ledande, så kommer en elektrisk ström att gå igenom saltlösningen när det elektriska fältet kopplas på. Den elektriska strömmen genom saltlösningen leder i sin tur till att lösningen värms upp. Risk finns alltså att saltlösningens temperatur stiger så mycket så att proteinerna upphör att fungera. Ett annat resultat av elektriska fält genom vattenbaserade lösningar är s.k. elektroosmotiskt flöde. Fenomenet elektroosmos innebär att lösningen försätts i en rörelse som är proportionell mot det elektriska fältets storlek. Vid kraftiga elektriska fält är det alltså möjligt att aktinet sköljs med i det elektroosmotiska flödet. Ytterligare en komplikation som kan uppstå vid elektriska fält genom IVMA-cellen är reaktioner i den beläggning som täcker glasytan vilken utgör botten på cellen. I detta examensarbete har en stor del av tiden ägnats åt att eliminera/reducera ovanstående oönskade bieffekter vid användandet av elektriska fält för att styra proteiner.</p>

Ph.D. afhandling, Roskilde universitetscenter 2007.

When simulating a molecular system, a cutoff distance for interactions is often used to speed up the simulations. This is made at the cost of neglecting some interactions which will lead to inaccurate results for energy, pressure components and surface tension (for systems with surfaces). To compensate for the neglected long-range interactions, continuum corrections can be added to the surface tension, system energies and pressures. For a homogenous isotropic system this is straight-forward but for a system with a surface it is more complicated. In this work we have derived expressions for the corrections to the surface tension, system energies and pressures that are more general than previous results. When these corrections are added to multi-component systems with a surface (or single-component systems with vacuum) they compensate for the change in surface tension, system energy and pressures due to the finite cutoff. When simulating systems with no Coulomb-interactions, the structure of the system may change significantly if the cutoffs are too short. If this is the case then these corrections alone will not be enough. The solution is to add corrections to the force acting on each molecule added during the simulation, which we derive in this work. This solves the structural problem at low cutoffs and makes it possible to calculate an accurate surface tension independent of cutoff.

The central theme in this thesis is reversibility. Two main attempts has been made to approach reversibility in cellular systems from both chemical and physical points of view. Reversibility of immunolabeling of proteins on the cell surface has been adressed by development of new fluorescent substances optimized for CALI (Chromophore-Assisted Laser Inactivation of protein). Aluminum phthalocyanine (AlPc) is here identified to be a good candidate for a new generation of fluorophores for efficient hydroxyl radical generation. It is shown that cells can be reversibly labeled with antibody-AlPc conjugates. In experiments on living cells the AlPcs were not only active as classic fluorophores but also as photocatalytic substances with destaining properties. Reversibility of cell immobilization is also reported, where cells cultured in microstructures were immobilized and 3D supported using hydrogels. Hydrogel formulation and application was optimized to achieve a system where both viability and ease of use was satisfied. Gel reversibility was actualized with pH and enzyme treatment. The developped method offers the possibility of stop flow culturing cells in controlled and reusable 3D environments. / <p>QC 20101102</p>

No description available.

The work presented in this thesis concerns the dynorphin neuropeptides, and dynorphin A (DynA) in particular. DynA belongs to the wider class of typical opioid peptides that, together with the opioid receptors, a four-membered family of GPCR membrane proteins, form the opioid system. This biological system is involved or implicated in several physiological processes such as analgesia, addiction and depression, and effects caused by DynA through this system, mainly through interaction with the kappa subtype of the opioid receptors (KOR), are called the opioid effects. In addition to this, non-opioid routes of action for DynA have been proposed, and earlier studies have shown that direct membrane interaction is likely to contribute to these non-opioid effects. The results discussed here fall into either of two categories; the interaction between DynA and a fragment of KOR, and the direct lipid interaction of DynA and two variant peptides.For the receptor interaction case, DynA most likely causes its physiological effects through binding its N-terminal into a transmembrane site of the receptor protein, while the extracellular regions of the protein, in particular the extracellular loop II (EL2), have been shown to be important for modulating the selectivity of KOR for DynA. Here we have focussed on the EL2, and show the feasibility of transferring this sequence into a soluble protein scaffold. Studies, predominantly by nuclear magnetic resonance (NMR) spectroscopy, of EL2 in this new environment show that the segment has the conformational freedom expected of a disordered loop sequence, while the scaffold keeps its native beta-barrel fold. NMR chemical shift and paramagnetic resonance enhancement experiments show that DynA binds with high specificity to EL2 with a dissociation constant of approximately 30 micro Molar, while binding to the free EL2 peptide is an order of magnitude weaker. The strength of these interactions are reasonable for a receptor recognition event. No binding to the naked scaffold protein is observed.In the second project, the molecules of interest were two DynA peptide variants recently found in humans and linked to a neurological disorder. Previously published reports from our group and collaborators pointed at very different membrane-perturbing properties for the two variants, and here we present the results of a follow-up study, where the variants R6W-DynA and L5S-DynA were studied by NMR and circular dichroism (CD) spectroscopy in solutions of fast-tumbling phospholipid bicelles, and compared with wild type DynA. Our results show that R6W-DynA interacts slightly stronger with lipids compared to wild type DynA, and much stronger compared to L5S-DynA, in terms of bicelle association, penetration and structure induction. These results are helpful for explaining the differences in toxicity, membrane perturbation and relationship to disease, between the studied neuropeptides.