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Hugonin, Loïc. "Spectroscopic studies of dynorphin neuropeptides and the amyloid beta-peptide : The consequences of biomembrane interactions." Doctoral thesis, Stockholm University, Department of Biochemistry and Biophysics, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-7156.
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<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>
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Liljesson, Kenneth. "Analys, elimination och reduktion av negativa bieffekter vid användning av elektriska fält i syfte att orientera molekylära motorer." Thesis, Växjö University, School of Mathematics and Systems Engineering, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:vxu:diva-2031.
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<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><br><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>
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Kowalewski, Jacob. "Modeling and Data Analysis in Cellular Biophysics." Doctoral thesis, KTH, Cellens fysik, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-11368.
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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.<br>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.<br>QC 20100726
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Olofsson, Per. "Microwell-based Collagen Matrix Migration Assays for NK:target Cell Interactions : Three-dimensional Imaging and Analysis of Transient Migration Behavior of NK Cells in vitro." Thesis, KTH, Cellens fysik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-43456.
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Natural killer (NK) cells are lymphocytes of the innate immune system responsible for lysing tumor and virally-infected cells. Investigating NK cell heterogeneity can inform the development of more efficacious immunotherapeutic treatments. Cell motility is an essential aspect of NK cell function. Moreover, the cell migration behavior within cell populations displays a marked heterogeneity. For some time it has been clear that cell-matrix interactions can radically alter the behavior of certain types of cells. (1) However, conventional studies of cell migration have relied on flat (2-D) surfaces, and thus do not take this potentially game-changing third dimension into account. Still, migration studies using ECM-mimicking biomaterials such as collagen and Matrigel may employ volume imaging, but often fail to quantify and analyze the vertical direction of migration. This project used silicon microchip-based technology, extracellular matrixlike type I collagen hydrogel, and fluorescence laser scanning confocal microscopy to study the migration behavior of single cells in 3-D. NK and targetcells were embedded in a collagen gel matrix deposited inside sub-mm scale microwells. The microwell provides natural barriers to cell migration, and so ensures that the cells remain confined within the imaging volume. The entire volume of the microwell was scanned for two hours by time-lapse fluorescence microscopy. A total of N = 14 NK cell migration trajectories were quantified using fluorescence centroid measurements. Results suggest that NK cells retain their cytotoxicity when embedded in the collagen matrix used for the 3-D migration assay. The average migration speed of the studied NK cells in three dimensions was found to be 3.7 ± 0.5μm/min (mean± SEM). Additionally, the NK cells exhibited a directional bias in migration, slightly preferring horizontal migration over vertical migration. In conclusion, this assay readily lends itself to short-term imaging of the migration behavior and cell-cell interactions of NK and target cells embedded in collagen gel in microwells. This microwell-gel system shows promising prospects for future applications at the interface of immunology and engineering.<br>NK-celler är lymfocyter tillhörande det ospecifika immunförsvaret vars uppgift är att uppsöka och avdöda tumör- och virusinfekterade celler. Genom att undersöka heterogeniteten inom NK-cellspopulationer öppnas en möjlighet att förbättra effektiviteten hos immunoterapeutiska behandlingar. Cellmotilitet är en viktig aspekt av NK-cellers funktion. Därutöver uppvisar cellmigrationsbeteendet inom cellpopulationer en märkbar heterogenitet. Det har under en tid stått klart att cell-matris-interaktioner kan ha en genomgripande effekt på beteendet hos vissa celltyper.(1) Emellertid grundar sig traditionella studier av cellmigration på användandet av tvådimensionella, plana ytor, och frånser på detta vis den potentiellt avgörande effekt som den tredje dimensionen kan ha på resultatet. Likväl kan studier som använder extracellulär matrix-liknande biomaterial, såsom kollagen och Matrigel, och som därutöver drar nytta av volymsavbildning för cellmigration ändå ofta bortse från att kvantifiera och analysera cellmigrationen i vertikalled. Detta projekt använde kiselbaserad mikrochipteknologi, extracellulär matrixliknande hydrogel typ I kollagen, samt fluorescensmikroskopi för att undersöka cellmigrationbeteendet hos enskilda NK-celler i 3-D. NK- och målceller bäddades in i en kollagenmatris vilken i sin tur gjöts in i en mindre än millimeterstor mikrobrunn. Mikrobrunnen utgör en naturlig barriär för cellmigration och kan således försäkra att cellerna stannar inuti avbildningsvolymen. Hela mikrobrunnens volym avbildades under två timmar med hjälp av tidsfördröjd fluorescensmikroskop. En tidsserie av mätningarna sammanställdes sedan. Totalt sammanställdes och kvantifierades N = 14 NK-cellers cellmigrationsbanor genom att uppskatta cellernas fluorescenta mittpunkter i den återskapade 3-D-volymen. Resultaten ger vid handen att NK-celler behåller sin cytotoxiska förmåga när de är inbäddade i 3-D-matrisen som används i mikrobrunnsuppsättningen. Den tredimensionella medelhastigheten för cellmigrationen hos de undersökta cellerna var 3.7±0.5 μm/min (medelvärde±standardfelet). Därutöver uppvisade NK-cellerna en bias i den genomsnittliga riktningen hos cellmigrationen, där horisontell cellmigration föredrogs framför vertikal cellmigration. Avslutningsvis kan sägas att denna experimentella uppsättning utan större problem kan användas för korttidsavbildning av cellmigrationsbeteende och cell-cell-interaktioner hos NK- och målceller inbäddade i en mikrobrunnsingjuten kollagenmatris. Detta mikrobrunn-gel-baserade system uppvisar lovande möjligheter för framtida tillämpningar i gränsytan mellan immunologi och ingenjörskonst.
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Meszaros, Johan. "Large area zone plate exposure by fixed beam moving stage lithography." Thesis, KTH, Biomedicinsk fysik och röntgenfysik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-44548.
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In this diploma project the so-called fixed beam moving stage (FBMS) module in the Raith 150 electron beam lithography system has been evaluated for making large area zone plate exposures. The project goal, besides the evaluation of the module, has been to find an exposure recipe for exposing zone plates with diameter up to 500 μm. The zone plates fabricated with this method will be used for synchrotron and x-ray free electron laser applications. The thesis starts with a short introduction to zone plate properties and fabrication procedures. Then the work where FBMS exposed zone plates are compared with normal write field exposures of 75 μm diameter zone plates is described. The conclusion is that for these small diameters, major problems with wobbly zones occur for the FBMS patterns. However, for larger diameters the pattern typically looks better. The final result with large area exposures are excellent zone plate patterns with 500 μm diameter and 100 nm outermost zone width. The total exposure time was 2 h 15 min. This relatively short time indicate that it will be practically possible to use the Raith system for these large area exposures.
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Oskar, Westlin. "Development of biophysical methods for characterization of PROTACs." Thesis, Uppsala universitet, Biokemi, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-455225.
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Targeting protein with the aim of ubiquitin-labelling for degradation is a new and upand coming field of drug discovery. Proteolysis targeting chimeras (PROTACs) areheterobifunctional linker molecules that connects the target molecule to an E3ubiquitin ligase, subsequent ubiquitination of the target protein initiates the proteindegradation by the proteosome system. The functional nature or mechanism ofPROTACs have the potential to reach previously undruggable proteins with shallowbinding pockets where traditionally designed inhibitors have failed. This projectexamines the possibility to develop a biophysical strategy for characterization andranking of PROTAC compounds. During the project biophysical methods such as SPRwith BIAcore T200, GCI with WAVE, switchSense and MST have been used todevelop a PROTAC ranking assay. Limited solubility of the PROTAC compoundshindered the development of a PROTAC ranking assay, the concentrations neededfor affinity estimation could not be reached due to the solubility of the PROTACcompounds under given condition. Hence further development of the PROTACranking assay is required. There is potentially a great deal of knowledge that can begathered from a working biophysical PROTAC ranking assay that can assist in thedevelopment of new therapeutic compounds.
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Akpe, Victor. "Photophysical and Chemical Approaches to Cellular Biophysics." Licentiate thesis, Stockholm : Fysik, Kungliga Tekniska högskolan, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-10097.
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Bagger, Heidi Louise. "Biophysical properties of proteins in solution : and the effects of protein glycosylation /." Roskilde : Department of Science, Systems and Models, Roskilde University, 2007. http://hdl.handle.net/1800/3322.
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Nordling, Torbjörn E. M. "Issues on modelling of large-scale cellular regulatory networks." Thesis, KTH, Numerical Analysis and Computer Science, NADA, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4182.
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<p>Vi har identifierat flexibelt utbyte och lagring av data i databaser, tillsammans med långvarig satsning på olika existerande och framtida modeller som nyckar till förståelse av det regler nätverk som utgör bron mellan geno- och fenotyp. Denna pilot studie av modellering av stora cellulära kontroll nätverk utgår från en intressant medicinsk frågeställning inom molekylär cellbiologi: Är framtvingad expression av Cdc6, aktivering av Cdk4/6 och Cdk2 tillräcklig för förankringsfri entré av cell cykelns S fas? Vi försöker konstruera en modell för att besvara denna fråga, på så sätt att vi kan detektera problem vid modellering av stora kontroll nätverk, diskutera implikationer och möjliga lösningar.</p><p>Vår modell är baserad på 1447 reaktioner och innehåller 1343 olika molekyler. Vi använde graf teori för att studera dess topologi och gjorde följande fynd: Nätverket är skalfritt och avtar enligt en potensfunktion, som var väntat baserat på tidigare arbeten. Nätverket består av ett stort väl förenat kluster. Det kan inte bli modulariserat i form av starka komponenter eller block i en användbar form. Detta eftersom vi fann en stor komponent eller ett stort block som innehöll majoriteten av alla molekyler och mer än hundra små komponenter eller block med en eller några molekyler. Vårt nätverk stämmer inte överens med en hierarkisk nätverks modell bestående av block förenade av cut-vertices.</p><br><p>We have identified flexible exchange and storage of data in databases, together with prolonged investment in different existing and future modelling formalisms as key issues in successful understanding of the regulatory network responsible for the connection between geno- and phenotype. This pilot study of modelling of large-scale regulatory networks starts with a medically interesting question from molecular cell biology: Is enforced expression of Cdc6, activation of Cdk4/6 and Cdk2 sufficient for anchorage-independent entry of the S phase of the cell cycle? We try to construct a model for answering this question, in such a way that we can reveal obstacles of large-scale regulatory modelling, discuss their implications and possible solutions.</p><p>Our model is based on 1447 reactions and contains 1343 different molecules. We used graph theory to study its topology and made the following findings: The network is scale-free and decays as a power-law, as could be expected based on earlier works. The network consists of one huge well connected cluster. It cannot be modularised into strong components or blocks in a useful way, since we get one big component or block containing a majority of all molecules and more than a hundred tiny components or blocks with one or a few molecules. Our network does not agree with a hierarchical network model consisting of blocks linked by cut-vertices.</p>
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Danielsson, Emma. "Towards a better understanding of protein structures : assessing the sulfur bridge in Cystine through photofragmentation." Thesis, Uppsala universitet, Materialteori, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-416437.
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This work aims to investigate the fragmentation of an ionized Cystine molecule, as simulated in the framework of molecular dynamics and quantum mechanics. Cystine is viewed as a model system for larger sets of peptides -- ultimately contributing to the understanding of protein photofragmentation, which is crucial for determining the structure of a protein using new methods. The analysis software was written in Python, partly in conjunction with another student. The photofragmentation of the molecule is analyzed in terms of bond integrity versus time and mass-to-charge ratios for the resulting fragments. Generally, the molecule disintegrates into more and smaller fragments the higher the degree of ionization is.<br>I det föreliggande arbetet undersöks fragmenteringen av en joniserad molekyl Cystin, som simulerats medelst molekyldynamik och kvantmekanik. Cystin betraktas som ett modellsystem för större peptidstrukturer -- något som i längden kan bidra till större förståelse för fotofragmentering av proteiner, vilket i sin tur är avgörande inom nya metoder för strukturbestämning. Analysprogrammet skrevs i Python och delvis i samarbete med en annan student. Molekylens fotofragmentering analyseras med avseende på bindningsintegritet över tid, samt mass-laddningskvot hos de resulterande fragmenten. I allmänhet sönderfaller molekylen till fler och mindre fragment ju högre joniseringsnivån är.
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Brian, Björn. "Microarray Technology for Kinetic Analysis of Vesicle Bound Receptor-Ligand Interactions." Thesis, Linköping University, The Department of Physics, Chemistry and Biology, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-8739.
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<p>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.</p>
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Liebau, Jobst. "Membrane interactions of glycosyltransferases." Licentiate thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-122485.
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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.
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Welin, Louise. "Påverkas Transversus Abdominis anticipatoriska aktivering av långvarig ihållande aktivering?" Thesis, Swedish School of Sport and Health Sciences, GIH, Department of Sport and Health Sciences, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:gih:diva-284.
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<p>Syfte</p><p>Studien syftade till att undersöka om långvarig ihållande submaximal aktivering av Transversus Abdominis (TrA) påverkar dess anticipatoriska aktivering vid snabba viljemässigt utförda armrörelser.</p><p>Metod</p><p>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.</p><p>Resultat</p><p>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.</p><p>Slutsats</p><p>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.</p><br><p>Aim</p><p>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.</p><p>Method</p><p>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.</p><p>Results</p><p>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.</p><p>Conclusion</p><p>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.</p>
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Myklatun, Ahne. "Production and Application of Micronsized Polysaccharide Particles - Studying Perturbation of a Model Mucus Barrier with Total Internal Reflection Fluorescence (TIRF) Microscopy and Atomic Force Microscopy (AFM) Indentation." Thesis, Norges teknisk-naturvitenskapelige universitet, Institutt for fysikk, 2011. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-13742.
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The overall aim of this project was to produce homogeneously sized polysaccharide microparticles and apply these and similar sized particles as probes for investigation of mucin layers as a model for a biological barrier. Small polysaccharide particles have many applications, e.g. within the medical field of drug delivery. In this study a microfluidic system was developed to produce alginate beads, which can be used in drug delivery systems. Different designs, continuous phases and concentrations were tested in order to find an optimal system. Beads in the size range of 10 µm were produced using a device with T-shaped design and three inlets. An electrostatic bead generator was also used to make alginate beads, however the beads produced were too large to be used in the experiments with the mucin layers.One of the many challenges when working with drug delivery systems is the mucus barrier protecting the epithelial cells. In this study a model mucus barrier was made by immobilizing mucins, the glycoprotein responsible for the physical properties of the barrier. A procedure for fluorescence labelling of polystyrene beads with quantum dots was developed, and penetration of these beads into the model barrier was measured with total internal reflection fluorescence (TIRF) microscopy. In TIRF the excitation field intensity decays exponentially, and the emitted fluorescence intensity from the beads gives an indication of the distance between the beads and the surface. Measurements performed on mucin layers of different concentrations indicate that mucin concentrations above 0,5 mg/ml will result in a layer too thick or too dense to give a intensity signal. At mucin concentration 0,05 mg/ml fluorescence was observable in TIRF, and it was clearly weaker than for the control with a bead directly on a glass surface. This indicates that the beads hover over the surface due to the mucin layers, and show that it is in principle possible to measure the penetration depth of beads into mucin layer using TIRF. To simulate the condition in the lungs of cystic fibrosis (CF) patients, the mucin layers were incubated with alginate. Measurements were performed to see how this affected the penetration of the beads into the layer. A weaker fluorescent signal was obtained for these samples in TIRF, which suggests that there has been interaction between mucin and alginate. It was in addition investigated how different concentrations of G-blocks in the solution affected the penetration into the mucin-alginate layer. These testes were carried out using both TIRF and atomic force microscopy (AFM) nanoindentation experiments. The TIRF measurements were inconclusive, while the nanoindentation experiments showed decreased interaction between mucin-alginate layer and a bead.
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Grøm, Vivian Aagesen. "UV-doser til psoriasispasienter og behandlingseffekt i løpet av tre ukers klimabehandling på Gran Canaria." Thesis, Norges teknisk-naturvitenskapelige universitet, Institutt for fysikk, 2011. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-13996.
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Psoriasis er en kronisk, inflammatorisk hudsykdom som gir tykke skjelldannelser på en rød hudoverflate. Sykdommen rammer 2-3% av den europeiske befolkningen. Ultrafiolett (UV) stråling som bruk i behandling av psoriasis er effektivt og naturlig. Solens UV-stråling vil forbedre de fleste av pasientenes psoriasisflekker. Samtidig som solen har en positiv effekt, vil overeksponering kunne forårsake direkte DNA-skade i hudcellene som igjen kan forårsake hudkreft. I den forbindelse vil det være viktig å finne mengden UV som vil gi størst klinisk utbytte for psoriasisflekkene, samtidig som den skader den omkringliggende friske huden minst mulig. Hypotesen bak, er at en optimal strålemengde også vil gi det beste kliniske resultatet. Oppgaven vil utforske UV-dose til 24 pasienter som deltok på en treukers klimabehandling på Gran Canaria i november 2010. Under oppholdet ble den maksimalt tilgjengelige UV-intensiteten målt kontinuerlig fra taket av behandlingssenteret. Dette ble samkjørt med at pasientene notert når og hvor lenge de hadde vært eksponert for solen i dagbøker. I tillegg bar hver pasient UV-dosimetre, ett på håndleddet og ett festet til badetøyet. Denne oppgaven skulle sammenstille de to målemetodene for å finne UV-dosen for hver pasients hud. Sammenligninger av de UV-doser funnet med de to metodene skulle også utføres og resultatene av målingene ble vurdert.Gjennomsnittlig individuell kumulativ dose for (målt med UVB-Biometeret i samsvar med dagbøkene) var på 216,5&#61617;27,8 SED. Målt individuell kumulativ gjennomsnittsdose (målt med dosimetrene) var på 107,2&#61617;25,6 SED og 63,5&#61617;21,6 SED for henholdsvis dosimeteret på håndleddet og det på badetøyet. Soleksponering i tre uker førte til en gjennomsnittlig Psoriasis Area and Severity Index (PASI)-reduksjon på 77,4&#61617;16,3%. Ingen signifikante verdier ble funnet som kan forutsi utfallet for psoriasispasientene. Det ble heller ikke påvist noen sammenheng mellom tid eksponert for solen og UV-dose eller dosen absorbert av huden i forhold til PASI- reduksjon.Dosimeteret festet til håndleddet fikk i gjennomsnitt 50% av den maksimalt tilgjengelige intensiteten. Den makstilgjengelige intensiteten vil måles for et horisontalt plan, og alle andre målinger med dosimetre på ulike områder av kroppen, kan sees på som en fraksjon av dette. Hvor på kroppen psoriasisflekken befinner seg vil ha mye å si for hvor høy UV-dose den faktisk vil få. Forskjellen mellom de ulike områdene vil være stor, i hovedsak grunnet ulik vinkling til forhold til solen. Dosen dosimetrene gir vil derfor bare være veiledende for hvor stor dose de ulike psoriasisflekkene faktisk fikk, avhengig av hvor på kroppen de var i forhold til dosimeteret.62,5% av pasientene oppnådde PASI75 (PASI-reduksjon&#61619;75%). Studien ble gjennomført i november som er en av månedene med lavest UV-indeks. Pasienter som får klimabehandling andre måneder av året med langt høyere UVI vil kunne få for høy eksponering for ultrafiolett stråling.
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Øyangen, Julia. "Photoprotection of riboflavin containing beverages." Thesis, Norges teknisk-naturvitenskapelige universitet, Institutt for fysikk, 2012. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-18386.
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Riboflavin, also known as vitamin B2 and one of the most easily absorbed nutrients,can be found in many different organisms. The most abundant source of riboflavin ismilk and dairy products; however it is also present in meat, fish and certain types ofvegetables and fruit. Riboflavin is an important part of a healthy diet in order to keep skin, eyes and nervous systems healthy. Some studies indicate that riboflavin plays an important role in cancer and cardiovascular diseases.As known, milk is extremely sensitive to light. Riboflavin is one of the factorsresponsible for the light-induced degradation of milk. In combination with light andoxygen riboflavin may act as a photosensitizer. When vitamin B2 absorbs blue-greenlight, an excited triplet state of riboflavin is generated through a process called intersystem crossing. Reactive oxygen species, such as singlet oxygen, is then formed by reaction of excited riboflavin triplet with dissolved oxygen present in milk. Light exposure of milk can lead to off-flavor and damage of vitamins by reaction of singlet oxygen with amino acids and lipids in milk. Unfortunately, most of the packaging materials today do not protect milk from light completely. The formation of singlet oxygen can also be prevented by adding quenchers that are able to deactivate riboflavin triplets.Certain amino acids and carotenoids are well known flavin quenchers.The purpose of this study was to investigate how well riboflavin triplets can bequenched by amino acids cysteine, histidine, methionine, tyrosine and tryptophan. Thequenching properties of hydrophilic carotenoid crocin were studied as well. Crocinhas been under investigation of researches at the Departement of Physics at NTNU.Lumiflavin, which is one of the riboflavin’s photodegradation products, was used instead of riboflavin. The former is more stable and has similar photochemical characteristics as the latter.The quenching of lumiflavin triplets was studied by using laser flash photolysis. Itconsists of irradiating the sample under investigation with a short-lived laser flash. The method was used to measure the kinetic decay rate of lumiflavin in aqueous buffer with and without different concentrations of a quencher. The data were fitted to two different decay models. From pseudo-first-order rate constants the quenching rate constants were determined for each amino acid and crocin. All amino acids and crocin used in this study showed a quenching effect on the lumiflavin triplets. Further, it was determined whether the fitting models are suitable for these kind of measurements by simulating the decay of lumiflavin with and without any quencher. More studies on the fitting models have to be done to be able to get reliable results.
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Sveinsson, Hrafn Mar. "Constrained Hydrogel swelling in Biological Sensors : A Finite Element Method Approach." Thesis, Norges teknisk-naturvitenskapelige universitet, Institutt for fysikk, 2012. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-19103.
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Material models has been developed for anionic and/or cationic hydrogels, with a simulation framework implemented in MATLAB and the finite element software ABAQUS. The geometry of the simulations is a hemispheroidal hydrogel, divided into a core with a shell, covalently attached to an optical fiber. The material models have been used to estimate the chemical parameters of poly-acrylamide hydrogels containing anionic or cationic monomer groups. Simulations comparing free and constrained swelling has been conducted in order to determine the effect of the geometrical constriction to the optical fiber. Constrained hydrogel swelling featuring shells with different properties than the core was also investigated.The aim of the study was to validate the material models and examine the effects of geometrical constrictions together with shell-impregnation. The anionic material model was shown to reproduce experimental swelling data, while the cationic material model only reproduced the data for ionic strength greater than 100 mM. Restricting the hydrogel to an optical fiber resulted in decreased change in volume and an increase in the axial swelling. The model was able to reproduce reported reduction in the swelling for an impregnated anionic hydrogel by using a neutral shell in the simulations, but failed to recreate the shape of the swelling curve. With the reduction of swelling as a basis, a new method for estimating thin-layer properties has been developed.
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Kiflemariam, Jordanos. "A Biomimetic Manganese Model for Artificial Photosynthesis : Q-band Electron Paramagnetic Resonance Study of a Novel Mn2(II,III) Complex." Thesis, Linköping University, Department of Science and Technology, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-4246.
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<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 ([Mn<sub>2</sub>(II,III)L(µ-OAc)<sub>2</sub>]ClO<sub>4</sub>), 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 Mn<sub>2</sub>(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, Mn<sub>2</sub>(bpmp)(µ-OAC)<sub>2</sub>. This is noticeable since Unsymmetric Mn2(II,III) and Mn<sub>2</sub> (bpmp)(µ-OAC)<sub>2</sub> 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>
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Olsson, Annakarin. "Piezoelectric Coatings on Implants : Sample preparation and construction of test-equipment for in vitro experiments." Thesis, Linköping University, The Department of Physics, Chemistry and Biology, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-4544.
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<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>
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Andersson, August. "The Application of isotropic bicelles as model membranes." Doctoral thesis, Stockholm University, Department of Biochemistry and Biophysics, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-497.
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<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 <sup>1</sup>H 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>
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Seibert, Mark Marvin. "Protein Folding and DNA Origami." Doctoral thesis, Uppsala universitet, Molekylär biofysik, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-121549.
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In this thesis, the folding process of the de novo designed polypeptide chignolin was elucidated through atomic-scale Molecular Dynamics (MD) computer simulations. In a series of long timescale and replica exchange MD simulations, chignolin’s folding and unfolding was observed numerous times and the native state was identified from the computed Gibbs free-energy landscape. The rate of the self-assembly process was predicted from the replica exchange data through a novel algorithm and the structural fluctuations of an enzyme, lysozyme, were analyzed. DNA’s structural flexibility was investigated through experimental structure determination methods in the liquid and gas phase. DNA nanostructures could be maintained in a flat geometry when attached to an electrostatically charged, atomically flat surface and imaged in solution with an Atomic Force Microscope. Free in solution under otherwise identical conditions, the origami exhibited substantial compaction, as revealed by small angle X-ray scattering. This condensation was even more extensive in the gas phase. Protein folding is highly reproducible. It can rapidly lead to a stable state, which undergoes moderate fluctuations, at least for small structures. DNA maintains extensive structural flexibility, even when folded into large DNA origami. One may reflect upon the functional roles of proteins and DNA as a consequence of their atomic-level structural flexibility. DNA, biology’s information carrier, is very flexible and malleable, adopting to ever new conformations. Proteins, nature’s machines, faithfully adopt highly reproducible shapes to perform life’s functions robotically.
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Hultin, Magnus. "Turnover of chylomicrons in the rat." Doctoral thesis, Umeå universitet, Institutionen för medicinsk kemi och biofysik, 1995. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-102338.
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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.<br><p>Diss. (sammanfattning) Umeå : Umeå universitet, 1995, härtill 5 uppsatser.</p><br>digitalisering@umu.se
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Carlsson, Gunilla. "Crystallography in Four Dimensions : Methods and Applications." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.bibl. [distributör], 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4301.
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von, Post Fredrik. "Microcontact printing of antibodies in complex with conjugated polyelectrolytes." Thesis, Linköping University, The Department of Physics, Chemistry and Biology, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-10123.
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<p>Microcontact printing using elastomeric stamps is a technique used in finding new and efficient ways to produce biodetection chips. Microcontact printed, with poly(dimetylslioxane) (PDMS) stamps, patterns of antibodies have been evaluated using fluorescence microscopy, imaging ellipsometry and atomic force microscopy. Fluorescent conjugated polyelectrolytes form non-covalent molecular complexes with Immunoglobulin-γ type antibodies, antigen binding to the tagged antibody result in spectroscopic shifts. Four different conjugated polyelectrolytes (POWT, POMT, PTT, PTAA) in complex with human serum albumin antibodies (aHSA) have been tested with fluorescence spectroscopy. Complexes of POWT and aHSA gave rise to thelargest wavelength shift when exposed to human serum albumin.</p><p>Several types of commercially available fluorescent antibodies and antigens were used to test the specificity of microcontact printed antibodies to different antigen solutions. Using fluorescence microscopy it could not be shown that printed antibody patterns promote specific adsorption of corresponding antigen. It is proposed however that changed surface characteristics of the substrate due to PDMS residues transferred during printing is the main driving force behind antigen adsorption.</p><p>POMT - poly (3-[(s)-5-amino-5-methoxylcarboxyl-3-oxapentyl]-2,5-thiophenylenehydrochloride)</p><p>POWT - poly (3-(s)-5-amino-5-carboxyl-3-oxapentyl]-2,5-thiophenylenehydrochloride)</p><p>PTAA - polytiophene acetic acid</p><p>PTT - poly (3-[2,5,8-trioxanonyl] thiophene)</p>
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Danielsson, Jens. "NMR studies of the amyloid beta-peptide." Doctoral thesis, Stockholm University, Department of Biochemistry and Biophysics, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-1410.
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<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>
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Österlund, Nicklas. "Gas phase studies of the Amyloid-β peptide : Peptide oligomerization and interactions with membrane mimetics". Thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-155009.
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The amyloid-β peptide is an amphiphilic peptide that exhibits self-aggregating properties, forming the amyloid fibrils that can be found in the brains of Alzheimer patients. Today it is believed that it is the soluble Aβ oligomers rather than the mature fibrils that are the main neurotoxic species. These small peptide assemblies are known to associate with lipid membranes and form pores that can transport Ca2+ into the cell, which in part could be responsible for their neurotoxic properties. However, most biophysical methods that have been developed to study Aβ target either the monomer or the mature fibrils, and not the low abundance and polydisperse oligomers. In this work, a soft ionization mass spectrometry method that retains non-covalent oligomer interactions in the gas phase has been developed. Using this method, monomeric and oligomeric Aβ (1-40) from dimers up to octamers, except heptamers, were detected in vitro. It was also possible to detect and study the effects of peptide modifications such as methionine-35 oxidation. As mass spectrometry is a non-averaging technique the aggregation kinetics for reduced and oxidized peptides are followed simultaneously, and the results showed that the oxidized form of Aβ(1-40) aggregates slower and forms lower amounts of soluble oligomers than the reduced form. Additionally, Aβ(1-40) interactions with zwitterionic SB3-14 detergent micelles were studied in the gas phase, and it was demonstrated that Aβ-micelle complexes can survive in the mass spectrometer and be identified. Detergent head group charges seem to be essential for peptide-micelle interaction, both in the gas phase and in solution as no structure induction is observed upon addition of the non-ionic detergent DDM. Overall gas phase and solution properties agree well, which is encouraging for future gas phase studies of Aβ interactions with membrane mimetics.
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Petzold, Katja. "NMR studies of host-pathogen interactions." Doctoral thesis, Umeå universitet, Medicinsk kemi och biofysik, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-25710.
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This thesis describes the use of Nuclear Magnetic Resonance (NMR) for characterizing two host-pathogen interactions: The behavior of a regulatory RNA of the Hepatitis B virus (HBV) and the attachment of Helicobacter pylori (H. pylori) to the gastric mucosa. NMR is a powerful tool in biomedicine, because molecules ranging from small ligands to biomacromolecules can be studied with atomic resolution. Different NMR experiments are designed to determine structures, or to monitor interactions, folding, stability or motion. Paper I describes the analysis of the motions of a regulatory RNA of HBV. The NMR structure of the RNA had revealed before that several well-conserved nucleotides adopt multiple conformations. Therefore an analysis of possible underlying motions was undertaken using two different NMR techniques, one of which (off-resonance ROESY) was applied to nucleic acids for the first time. The observed motions suggest an explanation why the structurally poorly defined nucleotides are highly conserved. In paper II we improved the ROESY NMR experiment, which is used to measure internuclear distances for structure determination of medium-sized molecules. Using a small protein and an organometallic complex as examples, we demonstrated that the new EASY ROESY experiment yields clean spectra that can directly be integrated to derive interatomic distances. H. pylori, the bacterium involved in peptic ulcer disease and gastric cancer, survives in the harsh acidic environment of the stomach. It possesses many membrane proteins which mediate adherence, raising the question, if their activity is related to membrane composition. In paper III & IV we analyzed therefore the phospholipid composition of H. pylori membranes. In paper III, an advanced method for the analysis of the phospholipid composition of biological membranes was developed. The two-dimensional semi-constant-time 31P,1H-COSY experiment combines information from phosphorus and hydrogen atoms of phospholipids for their unambiguous identification. Furthermore, the high resolution of the two-dimensional experiment allows the quantification of phospholipids where conventional methods fail. In paper IV we applied the new experiment to analyze the lipid composition of whole H. pylori cells, their inner and outer membranes, and of vesicles shed by the bacterium. The goal of this study was to characterize the vesicles which are suggested to play a role in the inflammation process. We established that the outer membrane and the vesicles have similar phospholipid compositions, suggesting that the vesicles are largely derived from the outer membrane. The NMR results presented here elucidate details of molecular systems engaged in pathogenicity, as basis for therapeutic strategies against these pathogens.
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Unnerståle, Sofia. "NMR Investigations of Peptide-Membrane Interactions, Modulation of Peptide-Lipid Interaction as a Switch in Signaling across the Lipid Bilayer." Licentiate thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-59534.
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The complexity of multi cellular organisms demands systems that facilitate communicationbetween cells. The neurons in our brains for instance are specialized in this cell-cellcommunication. The flow of ions, through their different ion channels, across the membrane, isresponsible for almost all of the communication between neurons in the brain by changing theneurons membrane potentials. Voltage-gated ion channels open when a certain thresholdpotential is reached. This change in membrane potential is detected by voltage-sensors in the ionchannels. In this licentiate thesis the Homo sapiens voltage- and calcium-gated BK potassiumchannel (HsapBK) has been studied. The NMR solution structure of the voltage-sensor ofHsapBK was solved to shed light upon the voltage-gating in these channels. Structures of othervoltage-gated potassium channels (Kv) have been determined by other groups, enablingcomparison among different types of Kv channels. Interestingly, the peptide-lipid interactions ofthe voltage-sensor in HsapBK are crucial for its mechanism of action.Uni cellular organisms need to sense their environment too, to be able to move towardsmore favorable areas and from less favorable ones, and to adapt their gene profiles to currentcircumstances. This is accomplished by the two-component system, comprising a sensor proteinand a response regulator. The sensor protein transfers signals across the membrane to thecytoplasm. Many sensor proteins contain a HAMP domain close to the membrane that isinvolved in transmitting the signal. The mechanism of this transfer is not yet revealed. Ourstudies show that HAMP domains can be divided into two groups based on the membraneinteraction of their AS1 segments. Further, these two groups are suggested to work by differentmechanisms; one membrane-dependent and one membrane-independent mechanism.Both the voltage-gating mechanism and the signal transduction carried out by HAMPdomains in the membrane-dependent group, demand peptide-lipid interactions that can be readilymodulated. This modulation enables movement of peptides within membranes or within thelipid-water interface. These conditions make these peptides especially suitable for NMR studies.
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Nilsson, Martin. "GIANT UNILAMELLAR VESICLES FOR PEPTIDE-MEMBRANE INTERACTION STUDIES USING FLUORESCENCE MICROSCOPY." Thesis, Linköpings universitet, Biofysik och bioteknik, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-167467.
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Vesicles are a type of biological or biomimetic particle consisting of one or more often spherical bilayers made up of amphipathic molecules, creating a closed system. They can function as an encapsulating device, holding hydrophilic molecules on the inside of the bilayer membrane(s) or hydrophobic molecules in the non-polar interstitial space in the middle of the bilayers. Because of this capacity to carry molecules, vesicles are a premier system for drug delivery and even theranostics in vivo. A peptide-based approach to release of encapsulated molecules has previously been developed but since drug delivery vesicles are in the size range of nanometers, the mechanisms have not been visualized. This project aims to produce giant unilamellar vesicles as a model system used to visualize membrane interactions vital to the understanding and further development of smaller vesicle-based systems for drug delivery. Giant unilamellar vesicles were produced successfully and a preparation protocol was established. Additionally, some membrane interactions were investigated using fluorescence microscopy.
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Unnersjö-Jess, David. "High-resolution imaging of kidney tissue samples." Licentiate thesis, KTH, Cellulär biofysik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-207577.
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The kidney is one of the most important and complex organs in the human body, filtering hundreds of litres of blood daily. Kidney disease is one of the fastest growing causes of death in the modern world, and this motivates extensive research for better understanding the function of the kidney in health and disease. Some of the most important cellular structures for blood filtration in the kidney are of very small dimensions (on the sub-200 nm scale), and thus electron microscopy has been the only method of choice to visualize these minute structures. In one study, we show for the first time that by combining optical clearing with STED microscopy, protein localizations in the slit diaphragm of the kidney, a structure around 75 nanometers in width, can now be resolved using light microscopy. In a second study, a novel sample preparation method, expansion microscopy, is utilized to physically expand kidney tissue samples. Expansion improves the effective resolution by a factor of 5, making it possible to resolve podocyte foot processes and the slit diaphragm using confocal microscopy. We also show that by combining expansion microscopy and STED microscopy, the effective resolution can be improved further. In a third study, influences on the development of the kidney were studied. There is substantial knowledge regarding what genes (growth factors, receptors etc.) are important for the normal morphogenesis of the kidney. Less is known regarding the physiology behind how paracrine factors are secreted and delivered in the developing kidney. By depleting calcium transients in explanted rat kidneys, we show that calcium is important for the branching morphogenesis of the ureteric tree. Further, the study shows that the calcium-dependent initiator of exocytosis, synaptotagmin, is expressed in the metanephric mesenchyme of the developing kidney, indicating that it could have a role in the secretion of paracrine growth factors, such as GDNF, to drive the branching.<br><p>QC 20170523</p>
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Papadopoulos, Evangelos. "Structural and functional studies of biomolecules with NMR and CD spectroscopy." Doctoral thesis, Stockholm University, Department of Biochemistry and Biophysics, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-7270.
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<p>Experimentally derived biomolecular structures were determined by Nuclear Magnetic Resonance (NMR). The properties of selected peptides and proteins in solution and in membrane mimicking micelles were observed by circular Dichroism (CD), mass spectrometry (MS), and other spectroscopic techniques.</p><p>The mDpl(1-30) peptide (30 residues) of the mouse Doppel protein was found to be positioned as an α-helix in a DHPC micelle. The same peptide can disrupt and cause leakage in small unilamellar vesicles.</p><p>Single D-amino acid isomers of Trp-cage (20 residues), the smallest peptide with a protein-like fold, were analyzed by CD spectroscopy and were found to have different secondary structures and melting temperatures. They were compared against MS measurements specially designed to reveal the secondary structure of proteins.</p><p>We studied a novel protein in E. coli of unknown structure that is encoded by the putative transcription factor ORF: ygiT (131 residues). This protein comprises a helix-turn-helix (HTH) domain in the C-terminus and contains two CxxC motives in the N-terminal domain, which binds Zn. This protein was named 2CxxC. We succeeded in overexpressing and purifying 2CxxC in E. coli with enough yield for a 13C, 15N uniformly labeled NMR sample. The chemical shift assignment was completed and the NMR structure was calculated in reducing, slightly acidic conditions (1mM DTT, pH 5.5). The determined HTH domain shows good similarity with structures predicted by a homology search, while the N-terminal domain has no other homologous structure in the Protein Data Bank (PDB).</p><p>The structure of the paddle region (27 residues) of the HsapBK(233-260) voltage and Ca+2 activated potassium channel, in DPC micelles, was determined by NMR. It shows a helix-turn-helix loop, which agrees well with the expected structure and could help to verify the proposed models of the voltage gating mechanism.</p><p>The C-repressor (dimer of 99 residues) of bacteriophage P2 was analyzed by NMR. We assigned the chemical shifts and NMR structure determination is under way.</p>
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Voevodskaya, Nina. "Paramagnetic states of diiron carboxylate proteins." Doctoral thesis, Stockholm : Department of Biochemistry and Biophysics, Stockholm University, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-652.
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Berntsson, Elina. "The effect of Edaravone on Amyloid beta aggregation." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-398469.
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Alzheimer’s disease (AD) is a devastating neurodegenerative disease that affect millions of people worldwide. Aggregation of Amyloid-β (Aβ) monomers create toxic oligomers that can interact with cellular membranes and disturb cellular functions, resulting in cell death and neurological dysfunction. Increased levels of oxidative stress have been shown in the brains of AD patients, something that besides the obvious cell and tissue toxicity, also favors the amyloidogenic pathway and generates more Aβ monomers. Here we show that Edaravone, a free radical scavenger can affect the aggregation rate of different lengths of Aβ. We show that Aβ-40 that is more commonly found in vivo aggregates faster with addition of Edaravone, while Aβ-42 aggregates slower or not at all. These findings add up to previous findings where free radical scavengers and antioxidants such as Edaravone have been suggested as a potential treatment in Alzheimer’s disease.
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Rane, Lukas. "Improving the temporal resolution of a microspectrometer for the study of the photophysics of enhanced green fluorescent protein." Thesis, KTH, Tillämpad fysik, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-300136.
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The use of fluorescent proteins as fluorescent markers has exploded over the last decades. In particular due to the development of advanced microscopy for live cell measurements, dynamic molecular studies down to single molecule levels and for superresolution microscopy. Many variants of fluorescent proteins exist with varying properties, such as emission color, photostability and brightness. These properties enable advanced applications, like timeresolved imaging or imaging below the diffraction limit. However, the photophysics of fluorescent proteins are complex and in many aspects quite unexplored. The triplet state in particular, is a central photophysical state because it is an entrance gate to an ensamble of deleterious photochemical processes that compromise the photostability of fluorescent proteins.The Pixel team at Institute de Biologie Structurale in France, is mainly focused on developing fluorescent proteins for advanced fluorescence imaging. One of the goals is to understand the influence of photochemistry on the properties of fluorescent proteins.In this project, a method to indirectly observe the triplet state in the prototypical EGFP fluorescent protein was developed. The introduction of new hardware and software, coupled to biophysical experiments, required an interdisciplinary strategy to tackle the obstacles during the route. Experiments under different environmental conditions to test the influence on the population of the triplet state of viscosity, pH, UV and infrared light, triplet state quenchers and temperature were performed.The results show that temperature and laser power greatly influence the triplet state kinetics in EGFP. Notably, it was found that the triplet state lifetime strongly increases at cryotemperature in comparison to roomtemperature. Overall, the newly developed setup and our preliminary results on EGFP open the door to novel studies on the photophysical properties of fluorescent proteins.<br>Nyttjandet av fluorescerande proteiner som markörer har exploderat de senaste årtionden. Speciellt till följd av utvecklingen av avancerad mikroskopi för levande cellmätningar, dynamiska molekylära studier ned till enstaka molekylnivåer och för superupplösnings mikroskopi. Många varianter av fluorescerande proteiner förekommer med varierande egenskaper så som färg, fotostabilitet och ljusstyrka. Dessa proteiner möjliggör avancerade applikationer, som tidsupplöst bildgivning eller bildgivning med upplösning under diffraktionsgränsen. Fotofysiken bakom fluorescerande proteiner är komplex och i många aspekter ganska outforskad. Triplettillståndet är ett centralt fotofysiskt tillstånd eftersom det är en ingångsport till en rad skadliga fotokemiska processer som äventyrar fotostabiliteten hos fluorescerance proteiner.Pixelteamet på Institute de Biologie Structurale i Frankrike, fokuserar huvudsakligen på utveckling av fluorescerande proteiner för avancerad fluorescerande bildgivning. Ett av målen är att förstå hur fotokemi påverkar egenskaperna hos fluorescerande proteiner.I det här projektet har en metod för att indirekt observera triplettillståndet i det prototypiska fluorescerande proteinet EGFP utvecklats. Introduktionen av ny hårdvara och mjukvara, i kombination med biofysikaliska experiment, krävde en interdisiplinär strategi för att tackla utmaningarna under vägens gång. Experiment under olika miljömässiga förhållanden gjordes för att testa hur populationen av triplettillståndet påverkas till följd av viskositet, pH, UV och infrarött ljus, triplettillståndshämmare och temperatur.Resultaten visar att temperatur och lasereffekt har en stor påverkan på triplettillståndet och dess kinetik hos EGFP. Noterbart är att triplettillståndets livstid ökar kraftigt i kryotemperatur i jämförelse med rumstemperatur. Sammanfattningsvis så utvecklades en ny experimentel uppställning och de tidiga resultaten från EGFP har öppnat dörren för nya studier rörande de fotofysiska egenskaperna hos fluorescerande proteiner.
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Viveca, Lindahl. "Optimizing sampling of important events in complex biomolecular systems." Doctoral thesis, KTH, Fysik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-217837.
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Proteins and DNA are large, complex molecules that carry out biological functions essential to all life. Their successful operation relies on adopting specific structures, stabilized by intra-molecular interactions between atoms. The spatial and temporal resolution required to study the mechanics of these molecules in full detail can only be obtained using computer simulations of molecular models. In a molecular dynamics simulation, a trajectory of the system is generated, which allows mapping out the states and dynamics of the molecule. However, the time and length scales characteristic of biological events are many orders of magnitude larger than the resolution needed to accurately describe the microscopic processes of the atoms. To overcome this problem, sampling methods have been developed that enhance the occurrence of rare but important events, which improves the statistics of simulation data. This thesis summarizes my work on developing the AWH method, an algorithm that adaptively optimizes sampling toward a target function and simultaneously finds and assigns probabilities to states of the simulated system. I have adapted AWH for use in molecular dynamics simulations. In doing so, I investigated the convergence of the method as a function of its input parameters and improved the robustness of the method. I have also worked on a generally applicable approach for calculating the target function in an automatic and non-arbitrary way. Traditionally, the target is set in an ad hoc way, while now sampling can be improved by 50% or more without extra effort. I have also used AWH to improve sampling in two biologically relevant applications. In one paper, we study the opening of a DNA base pair, which due to the stability of the DNA double helix only very rarely occurs spontaneously. We show that the probability of opening depends on both nearest-neighbor and longer-range sequence effect and furthermore structurally characterize the open states. In the second application the permeability and ammonia selectivity of the membrane protein aquaporin is investigated and we show that these functions are sensitive to specific mutations.<br><p>QC 20171117</p>
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Bernhem, Kristoffer. "Quantitative bioimaging in single cell signaling." Doctoral thesis, KTH, Tillämpad fysik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-215076.
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Imaging of cellular samples has for several hundred years been a way for scientists to investigate biological systems. With the discovery of immunofluorescence labeling in the 1940’s and later genetic fluorescent protein labeling in the 1980’s the most important part in imaging, contrast and specificity, was drastically improved. Eversince, we have seen a increased use of fluorescence imaging in biological research, and the application and tools are constantly being developed further. Specific ion imaging has long been a way to discern signaling events in cell systems. Through use of fluorescent ion reporters, ionic concentrations can be measured inliving cells as result of applied stimuli. Using Ca2+ imaging we have demonstrated that there is a inverse influence by plasma membrane voltage gated calcium channels on angiotensin II type 1 receptor (a protein involved in blood pressure regulation). This has direct implications in treatment of hypertension (high blood pressure),one of the most common serious diseases in the western civilization today with approximately one billion afflicted adults world wide in 2016. Extending from this more lower resolution live cell bioimaging I have moved into super resolution imaging. This thesis includes works on the interpretation of super resolution imaging data of the neuronal Na+, K+ - ATPase α3, a receptor responsible for maintaining cell homeostasis during brain activity. The imaging data is correlated with electrophysiological measurements and computer models to point towards possible artefacts in super resolution imaging that needs to be taken into account when interpreting imaging data. Moreover, I proceeded to develop a software for single-molecule localization microscopy analysis aimed for the wider research community and employ this software to identify expression artifacts in transiently transfected cell systems. In the concluding work super-resultion imaging was used to map out the early steps of the intrinsic apoptotic signaling cascade in space and time. Using superresoultion imaging, I mapped out in intact cells at which time points and at which locations the various proteins involved in apoptotic regulation are activated and interact.<br>Avbildning av biologiska prover har i flera hundra år varit ett sätt för forskare att undersöka biologiska system. Med utvecklingen av immunofluoresens inmärkn-ing och fluoresens-mikroskopi förbättrades de viktigaste aspekterna av mikroskopi,kontrast och specificitet. Sedan 1941 har vi sett kontinuerligt mer mångsidigt och frekvent användning av fluorosense-mikroskopi i biologisk forskning. Jon-mikroskopi har länge varit en metod att studera signalering i cell-system. Genom användning av fluorosenta jon-sensorer går det att mäta variationer avjon koncentrationer i levande celler som resultat av yttre påverkan. Genom att använda Ca2+ mikroskopi har jag visat att det finns en omvänd koppling mellan kalcium-kanaler i plasma-membran och angiotensin II typ 1 receptorn (ett proteininvolverat i blodtrycksreglering). Detta har direkta implikationer för behandlingav högt blodtryck, en av de mer vanliga sjukdomarna i västvärlden idag med överen miljard drabbade patienter i världen 2016. Efter detta projekt vidgades mitt fokus till att inkludera superupplösnings-mikroskopi. Denna avhandling inkluderar ett arbete fokuserat på tolkningen av superupplösnings-mikroskopi data från neuronal Na+, K+ - ATPase α3, en jon-pump som återställer cellernas jonbalans i samband med cell signalering. Mikroskopi-datan korreleras mot elektrofysiologi experiment och modeller för att illustrera möjliga artefakter i superupplösnings-mikroskopi som måste tas i beaktande i samband med tolkning av data. Jag fortsatte med att utveckla mjukvara för analys av data från singel-molekyl-lokalisations-mikroskopi där fokuset för mjukvaran framförallt varit på användarvänligheten. Detta då jag hoppas att den kommer vara användbar för ett bredare forskingsfält. Mjukvaran användes även i ett separat projekt för att identifiera överuttrycks-artefakter i transfekterade celler. I det avslutande arbetet använder jag superupplösnings-mikroskopi för att karakterisera de tidiga stegen i mitokondriell apoptos. Jag identifierar när och var i cellen de olika proteinerna involverade i apoptos signaleringen är aktiverade och interagerar.<br><p>QC 20171003</p>
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Westerlund, Annie M. "Computational Study of Calmodulin’s Ca2+-dependent Conformational Ensembles." Licentiate thesis, KTH, Biofysik, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-234888.
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Ca2+ and calmodulin play important roles in many physiologically crucial pathways. The conformational landscape of calmodulin is intriguing. Conformational changes allow for binding target-proteins, while binding Ca2+ yields population shifts within the landscape. Thus, target-proteins become Ca2+-sensitive upon calmodulin binding. Calmodulin regulates more than 300 target-proteins, and mutations are linked to lethal disorders. The mechanisms underlying Ca2+ and target-protein binding are complex and pose interesting questions. Such questions are typically addressed with experiments which fail to provide simultaneous molecular and dynamics insights. In this thesis, questions on binding mechanisms are probed with molecular dynamics simulations together with tailored unsupervised learning and data analysis. In Paper 1, a free energy landscape estimator based on Gaussian mixture models with cross-validation was developed and used to evaluate the efficiency of regular molecular dynamics compared to temperature-enhanced molecular dynamics. This comparison revealed interesting properties of the free energy landscapes, highlighting different behaviors of the Ca2+-bound and unbound calmodulin conformational ensembles. In Paper 2, spectral clustering was used to shed light on Ca2+ and target protein binding. With these tools, it was possible to characterize differences in target-protein binding depending on Ca2+-state as well as N-terminal or C-terminal lobe binding. This work invites data-driven analysis into the field of biomolecule molecular dynamics, provides further insight into calmodulin’s Ca2+ and targetprotein binding, and serves as a stepping-stone towards a complete understanding of calmodulin’s Ca2+-dependent conformational ensembles.<br><p>QC 20180912</p>
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Magnusson, Karin. "DNA chips with conjugated polyelectrolytes as fluorophore in fluorescence amplification mode." Thesis, Linköping University, The Department of Physics, Chemistry and Biology, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-11559.
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<p>The aim of this diploma work is to improve selectivity and sensitivity in DNA-chips by utilizing fluorescence resonance energy transfer (FRET) between conjugated polyelectrolytes (CPEs) and fluorophores.</p><p>Leclerc and co-workers have presented successful results from studies of super FRET between fluorophore tagged DNA and a CPE during hybridisation of the double strand. Orwar and co-workers have constructed a DNA-chip using standard photo lithography creating a pattern of the hydrophobic photoresist SU-8 and cholesterol tagged DNA (chol-DNA). This diploma work will combine and modify these two ideas to fabricate a improved DNA-chip.</p><p>Immobilizing of DNA onto surface has been done by using soft lithography. Hydrophobic pattern arises from the poly(dimethylsiloxane) (PDMS) stamp. The hydrophobic pattern will attract chol-DNA that is adsorbed to the chip. Different sets of fluorophores are covalently bound to the DNA and adding CPEs to the complex will make FRET occur between CPE and bound fluorophore.</p><p>We will here show that the specificity in DNA hybridization by using PDMS patterning was high. FRET clearly occurred, especially with the CPEs as donor to the fluorophore Cy5. The intensity of FRET was higher when the fluorophore and the CPE were conjugated to the same DNA strand. The largest difference in FRET intensity between double stranded and single stranded complexes was observed with the CPE tPOMT. Super FRET has been observed but not yet fully proved. The FRET efficiency was lower with the fluorophore Alexa350 as donor compared to the Cy5/CPE complex. Most of the energy transferred from Alexa350 was extinguished by quenching.</p>
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Oglęcka, Kamila. "Biophysical studies of membrane interacting peptides derived from viral and Prion proteins." Doctoral thesis, Stockholm University, Department of Biochemistry and Biophysics, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-7109.
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<p>This thesis focuses on peptides derived from the Prion, Doppel and Influenza haemagglutinin proteins in the context of bilayer interactions with model membranes and live cells. The studies involve spectroscopic techniques like fluorescence, fluorescence correlation spectroscopy (FCS), circular and linear dichroism (CD and LD), confocal fluorescence microscopy and NMR.</p><p>The peptides derived from the Prion and Doppel proteins combined with their subsequent nuclear localization-like sequences, makes them resemble cell-penetrating peptides (CPPs). mPrPp(1-28), corresponding to the first 28 amino acids of the mouse PrP, was shown to translocate across cell membranes, concomitantly causing cell toxicity. Its bovine counterpart bPrPp(1-30) was demonstrated to enter live cells, with and without cargo, mainly via macropinocytosis. The mPrPp(23-50) peptide sequence overlaps with mPrPp(1-28) sharing the KKRPKP sequence believed to encompass the driving force behind translocation. mPrPp(23-50) was however found unable to cross over cell membranes and had virtually no perturbing effects on membranes.</p><p>mDplp(1-30), corresponding of the first 30 N-terminal amino acids of the Doppel protein, was demonstrated to be almost as membrane perturbing as melittin. NMR experiments in bicelles implied a transmembrane configuration of its alpha-helix, which was corroborated by LD in vesicle bilayers. The positioning of the induced alpha-helix in transportan was found to be more parallel to the bilayer surface in the same model system.</p><p>Positioning of the native Influenza derived fusion peptide in bilayers showed no pH dependence. The glutamic acid enriched variant however, changed its insertion angle from 70 deg to a magic angle alignment relative the membrane normal upon a pH drop from 7.4 to 5.0. Concomitantly, the alpha-helical content dramatically rose from 18% to 52% in partly anionic membranes, while the native peptide’s helicity increased only from 39% to 44% in the same conditions.</p>
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Klenkar, Goran. "Protein Microarray Chips." Doctoral thesis, Linköping : Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-8904.
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Zamotin, Vladimir. "Structural studies of heterogeneous amyloid species of lysozymes and de novo protein albebetin and their cytotoxicity." Doctoral thesis, Umeå : Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1085.
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Bergh, Magnus. "Interaction of Ultrashort X-ray Pulses with Material." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis Acta Universitatis Upsaliensis, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8274.
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Karjalainen, Eeva-Liisa. "The choreography of protein vibrations : Improved methods of observing and simulating the infrared absorption of proteins." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-60415.
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The work presented in this thesis has striven toward improving the capability to study proteins using infrared (IR) spectroscopy. This includes development of new and improved experimental and theoretical methods to selectively observe and simulate protein vibrations. A new experimental method of utilising adenylate kinase and apyrase as helper enzymes to alter the nucleotide composition and to perform isotope exchange in IR samples was developed. This method enhances the capability of IR spectroscopy by enabling increased duration of measurement time, making experiments more repeatable and allowing investigation of partial reactions and selected frequencies otherwise difficult to observe. The helper enzyme mediated isotope exchange allowed selective observation of the vibrations of the catalytically important phosphate group in a nucleotide dependent protein such as the sarcoplasmic reticulum Ca2+-ATPase. This important and representative member of P-type ATPases was further investigated in a different study, where a pathway for the protons countertransported in the Ca2+-ATPase reaction cycle was proposed based on theoretical considerations. The transport mechanism was suggested to involve separate pathways for the ions and the protons. Simulation of the IR amide I band of proteins enables and supports structure-spectra correlations. The characteristic stacking of beta-sheets observed in amyloid structures was shown to induce a band shift in IR spectra based on simulations of the amide I band. The challenge of simulating protein spectra in aqueous medium was also addressed in a novel approach where optimisation of simulated spectra of a large set of protein structures to their corresponding experimental spectra was performed. Thereby, parameters describing the most important effects on the amide I band for proteins could be determined. The protein spectra predicted using the optimised parameters were found to be well in agreement with experiment.<br><p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Manuscript.</p>
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Liebau, Jobst. "Taming the Griffin : Membrane interactions of peripheral and monotopic glycosyltransferases and dynamics of bacterial and plant lipids in bicelles." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-146872.
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Biological membranes form a protective barrier around cells and cellular compartments. A broad range of biochemical processes occur in or at membranes demonstrating that they are not only of structural but also of functional importance. One important class of membrane proteins are membrane-associated glycosyltransferases. WaaG is a representative of this class of proteins; its function is to catalyze one step in the synthesis of lipopolysaccharides, which are outer membrane lipids found in Gram-negative bacteria. To study protein-membrane complexes by biophysical methods, one must employ membrane mimetics, i.e. simplifications of natural membranes. One type of membrane mimetic often employed in solution-state NMR is small isotropic bicelles, obloid aggregates formed from a lipid bilayer that is dissolved in aqueous solvent by detergent molecules that make up the rim of the bicelle. In this thesis, fast dynamics of lipid atoms in bicelles containing lipid mixtures that faithfully mimic plant and bacterial membranes were investigated by NMR relaxation. Lipids were observed to undergo a broad range of motions; while the glycerol backbone was found to be rigid, dynamics in the acyl chains were much more rapid and unrestricted. Furthermore, by employing paramagnetic relaxation enhancements an ‘atomic ruler’ was developed that allows for measurement of the immersion depths of lipid carbon atoms. WaaG is a membrane-associated protein that adopts a GT-B fold. For proteins of this type, it has been speculated that the N-terminal domain anchors tightly to the membrane via electrostatic interactions, while the anchoring of the C-terminal domain is weaker. Here, this model was tested for WaaG. It was found by a set of circular dichroism, fluorescence, and NMR techniques that an anchoring segment located in the N-terminal domain termed MIR-WaaG binds electrostatically to membranes, and the structure and localization of isolated MIR-WaaG inside micelles was determined. Full-length WaaG was also found to bind membranes electrostatically. It senses the surface charge density of the membrane whilst not discriminating between anionic lipid species. Motion of the C-terminal domain could not be observed under the experimental conditions used here. Lastly, the affinity of WaaG to membranes is lower than expected, indicating that WaaG should not be classified as a monotopic membrane protein but rather as a peripheral one.<br><p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Manuscript.</p>
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Rogvall, Johanna. "The sensitivity of the EMC algorithm to the light intensity and amount of diffraction patterns in diffraction experiments." Thesis, Uppsala universitet, Institutionen för fysik och astronomi, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-448777.
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To understand the function of macromolecules like proteins it helps to know the structure of the molecule. Coherent diffraction imaging is an emerging method that might be used to figure out the structures of macromolecules. In this method diffraction patterns of the macromolecule are recorded by shining light on the molecule from many unknown orientations and detecting the pattern of the diffracted photons. By assembling the diffraction patterns in a specific way and finding the phase of the photons that gave rise to the diffraction patterns, it is theoretically possible to obtain the electronstructure of the molecule and thus the molecular structure. The assembling of several thousand diffraction patterns representing unknown orientations of the molecule is hard to do by hand, but there are several methods that can be used. The EMC (Expand-Maximize-Compress) algorithm is one of those methods. It is an iterative algorithm that tries to create a model describing the Fourier Transform of the electron density of the molecule by maximizing each diffraction patterns fit to the model. This work examines how sensitive the EMC algorithm is to datasets with few diffraction patterns or a low intensity of the light being diffracted by the molecule, for the proteins phytochrome and lysozyme. The result of the work could be used to make sure enough data in collected in real experiments. Diffraction patterns simulated with the program Condor is used in this work, instead of diffraction patterns from real experiments.EMC finds the correct model when the data set contains about 1/3 fewer photons for the smaller more symmetrical molecule lysozyme than it does for phytochrome. This might be because the shapes in lysozymes diffraction patterns are larger than in phyochrome’s patterns. For phytochrome the EMC algorithm assembled the diffraction patterns correctly, with fewest photons for the light intensity 0.764 J/μm2 and 1250 diffraction patterns. For lysozyme it was with an intensity 1.910 J/μm2 and 1425 diffraction patterns. More investigation of the data is needed to understand what factors that affect the EMC algorithms ability to assemble the diffraction patterns correctly.<br>För att förstå makromolekylers kemiska eller biologiska funktion so underlättar det om man känner till molekylens kemiska struktur. Med den nya tekniken “coherent diffraction imaging” ska det vara möjligt att lista ut makromolekylers struktur. I denna teknik detekterar man diffraktionsmönster av molekylen genom att belysa molekylen med ljus från många olika okända vinklar and registrera mönstret som skapas av det diffrakterade ljuset. Genom att sätta ihop alla dessa diffraktionsmönster på rätt sätt och sen återskapa fasen för ljuset i diffraktionsmönstret så kan man generera molekylens elektronstruktur och från elektronstrukturen kan man få tag i molekylens struktur. Att sätta ihop tio tusentals diffraktionsmönster med okända vinklar på rätt sätt är väldigt svårt att göra, men det finns flera olika metoder som kan användas. EMC (Expand-Maximize-Compress) är en sådan metod. EMC är en iterativ algoritm som skapar en modell av (Fourier transformen av) molekylens elektronstruktur genom att maximera hur bra diffraktionsmönstren passar med modellen. Detta arbete utreder hur bra EMC algoritmen är på att hitta rätt (Fourier transform av) elektronstruktur när väldigt få diffraktionsmönster används eller när intensiteten på ljuset som sprids av molekylen är lågt. Programmet Condor används för att generera teoretiska diffraktionsmönster för de 2 molekylerna lysozym och fytokrom. EMC används sedan med olika uppsättningar av intensitet och antal diffraktionsmönster för att skapa en modell av elektronstrukturen. EMC behövde ca 1/3 färre antal fotoner i sin modell för att hittar den rätta modellen av elektronstrukturen för den lilla symmetriskt formade molekylen lysozym än för fytokrom. Att det är lättare för EMC algoritmen att hitta den korrekta modellen för lysozym än fytokrom kan bero på att lysozyms diffraktionsmönster har större former/features eller på lysozyms storlek och form. EMC körningen som behövde minst antal fotoner för att hitta den korrekta elektronstrukturen för fytokrom hade intensiteten 0,764 J/μm2 på det inkommande ljuset och behövde 1250 diffraktionsmönster. För lysozym behövdes det 1,910 J/μm2 och 1425 diffraktionsmönster för att EMC algoritmen skulle hitta rätt modell av elektronstrukturen.
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Gutheim, Sabina. "Characterization of Alcohol Modulation of a Pentameric Ligand-gated Ion Channel with Electrophysiology and Molecular Dynamics Simulations." Thesis, KTH, Tillämpad fysik, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-296530.
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Pentameric ligand-gated ion channels (pLGICs) are membrane receptors that play a crucial role in every living organism. The pLGIC protein structure forms a pore through the membrane of a cell that can let specific ions pass through, upon activation by endogenous agonists. pLGICs are allosterically modulated by ligands binding at allosteric sites, that either stabilize a certain conformation or change the binding affinity of the endogenous agonist. However, much remains unknown about the exact way in which these modulators bind to and affect pLGICs. An increased understanding could help in the search for novel and/or more effective target drugs. With this masters thesis, I hope to contribute by investigating the modulatory effect of ethanol on the bacterial Gloeobacter ligand-gated ion channel (GLIC). This has been done by performing oocyte electrophysiology recordings and analysis of molecular dynamics simulations, both with and without ethanol, and of four separate variants of GLIC that are either potentiated or inhibited by ethanol. Two possible allosteric sites were discovered in a transmembraneintrasubunit pocket: a potentiating allosteric site close to the M2 helix and residue V242, as well as an inhibitory membrane- and M4 helix-close intrasubunit site. Finally, evidence was found that could support a previously suggested inhibitory allosteric site in the pore around the 9’ hydrophobic gate.<br>Pentameriska ligandstyrda jonkanaler (pLGICs) är membranreceptorer som utgör vitala delar av varje levande organism. pLGICs proteinstruktur formar en por genom cellmembranet, som kan släppa igenom specifika joner efter aktivering av endogena agonister. pLGICs är allostermodulerade av ligander som binder vid allostera säten och som därigenom antingen stabiliserar en viss form eller förändrar den endogena agonistens bindningsstyrka. Emellertid saknas fortfarande mycket kunskap på detaljnivå om hur dessa modulatorer binder sig till och påverkar kanalerna. En ökad förståelse skulle hjälpa forskningen efter nya och/eller mer effektiva mediciner. Mitt examensarbetehoppas bidra genom att studera hur etanol modulerar den bakteriella ligandstyrda jonkanalen GLIC från Gloeobacter. Det har gjorts genom elektrofysiologimätningar på oocyter och analys av molekulärdynamiksimuleringar, båda av fyra olika GLIC-varianter, som antingen potentieras eller hämmas av etanol, och med eller utan etanol. Två allostera säten upptäcktes i det transmembrana intrasubenhetområdet: ett säte för potentiering nära M2 helixen och aminosyran V242, och ett hämmande säte nära membranet och helix M4. Slutligen hittades tecken som kan styrka existensen av det tidigare föreslagna hämmande allostera sätet i poren kring den hydrophoba porten.
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Lepp, Håkan. "Experimental studies of proton translocation reactions in biological systems : Electrogenic events in heme-copper oxidases." Doctoral thesis, Stockholm University, Department of Biochemistry and Biophysics, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-8147.
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<p>Terminal heme-copper oxidases (HCuOs) are transmembrane proteins that catalyze the final step in the respiratory chain - the reduction of O<sub>2</sub> to H<sub>2</sub>O, coupled to energy conservation by generation of an electrochemical proton gradient. The most extensively investigated of the HCuOs are the <i>aa</i><sub>3</sub>-type oxidases, to which cytochrome <i>c</i> oxidase (Cyt<i>c</i>O) belongs, which uses energy released in the O<sub>2</sub>-reduction for proton pumping. The bacterial nitric oxide reductases (NORs) have been identified as divergent members of the HCuO-superfamily and are involved in the denitrification pathway where they catalyze the reduction of NO to NO<sub>2</sub>. Although as exergonic as O<sub>2</sub>-reduction, this reaction is completely non-electrogenic. Among the traditional HCuOs, the <i>cbb</i><sub>3</sub>-type oxidases are the closest relatives to the NORs and as such provide a link between the <i>aa</i><sub>3</sub> oxidases and the NORs. The <i>cbb</i><sub>3</sub> oxidases have been shown to pump protons with nearly the same efficiency as the <i>aa</i><sub>3</sub> oxidases, despite low sequence similarity.</p><p>This thesis is focused on measurements of membrane potential generating reactions during catalysis in the Cyt<i>c</i>O and the <i>cbb</i><sub>3</sub> oxidase from <i>Rhodobacter sphaeroides</i>, and the NOR from <i>Paracoccus</i> <i>denitrificans</i>, using a time resolved electrometric technique. The pH dependence of the membrane potential generation in Cyt<i>c</i>O showed that only one proton is taken up and that no protons are pumped, at high pH. An additional kinetic phase was also detected at high pH that presumably originates to from charge-transfer within the K-pathway. Possible reasons for uncoupling, and the extent of charge-transfer, were studied using structural variants of Cyt<i>c</i>O. The measurements established that electrons and protons are taken up from the same side of the membrane in NOR. In addition, the directionality for proton uptake in <i>cbb</i><sub>3</sub> oxidase appeared to be dependent on the choice of substrate while proton pumping was indicated to occur only during O<sub>2</sub>-reduction.</p>
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Sani, Marc-Antoine. "Apoptosis Regulation via the Mitochondrial Pathway : Membrane Response upon Apoptotic Stimuli." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1883.
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The aim of this thesis was the investigation of the mitochondrial response mechanisms upon apoptotic stimuli. The specific objectives were the biophysical characterization of membrane dynamics and the specific roles of lipids in the context of apoptotic regulation occurring at the mitochondrion and its complex membrane systems. The BH4 domain is an anti-apoptotic specific domain of the Bcl-2 protein. Solid phase peptide synthesis was used to produce large amount of the peptide for biophysical studies. A protocol has been established and optimized, guarantying the required purity for biophysical studies. In detail the purification by high performance liquid chromatography and the characterisation via mass spectroscopy are described. The secondary structure of BH4 changes significantly in the presence of lipid vesicles as observed by infrared spectroscopy and circular dichroism. The BH4 peptide aggregates at the membrane surface and inserts slightly into the hydrophobic part of the membrane. Using nuclear magnetic resonance (NMR) and calorimetry techniques, it could even be shown that the BH4 domain modifies the dynamic and organization of the liposomes which mimic a mitochondrial surface. The second study was on the first helix of the pro-apoptotic protein Bax. This sequence called Bax-α1 has the function to address the cytosolic Bax protein to the mitochondrial membrane upon activation. Once again a protocol has been established for the synthesis and purification of this peptide. The aim was to elucidate the key role of cardiolipin, a mitochondria-specific phospholipid, in the interaction of Bax-α1 with the mitochondrial membrane system. The NMR and circular dichroism studies showed that Bax-α1 interacts with the membrane models only if they contain the cardiolipin, producing a strong electrostatic lock effect which is located at the membrane surface. Finally, a new NMR approach was developed which allows the investigation of the lipid response of isolated active mitochondria upon the presence of apoptotic stimuli. The goal was there to directly monitor lipid specific the occurring changes during these physiological activities.
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Marklund, Emil. "Bayesian inference in aggregated hidden Markov models." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-243090.
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Single molecule experiments study the kinetics of molecular biological systems. Many such studies generate data that can be described by aggregated hidden Markov models, whereby there is a need of doing inference on such data and models. In this study, model selection in aggregated Hidden Markov models was performed with a criterion of maximum Bayesian evidence. Variational Bayes inference was seen to underestimate the evidence for aggregated model fits. Estimation of the evidence integral by brute force Monte Carlo integration theoretically always converges to the correct value, but it converges in far from tractable time. Nested sampling is a promising method for solving this problem by doing faster Monte Carlo integration, but it was here seen to have difficulties generating uncorrelated samples.
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Cardoch, Sebastian. "Computational study of single protein sensing using nanopores." Thesis, Uppsala universitet, Materialteori, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-423441.
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Identifying the protein content in a cell in a fast and reliable manner has become a relevant goal in the field of proteomics. This thesis computationally explores the potential for silicon nitride nanopores to sense and distinguish single miniproteins, which are small domains that promise to facilitate the systematic study of larger proteins. Sensing and identification of these biomolecules using nanopores happens by studying modulations in ionic current during translocation. The approach taken in this work was to study two miniproteins of similar geometry, using a cylindrical-shaped pore. I employed molecular mechanics to compare occupied pore currents computed based on the trajectory of ions. I further used density functional theory along with relative surface accessibility values to compute changes in interaction energies for single amino acids and obtain relative dwell times. While the protein remained inside the nanopore, I found no noticeable differences in the occupied pore currents of the two miniproteins for systems subject to 0.5 and 1.0 V bias voltages. Dwell times were estimated based on the translocation time of a protein that exhibits no interaction with the pore walls. I found that both miniproteins feel an attractive force to the pore wall and estimated their relative dwell times to differ by one order of magnitude. This means even in cases where two miniproteins are indistinguishable by magnitude changes in the ionic current, the dwell time might still be used to identify them. This work was an initial investigation that can be further developed to increase the accuracy of the results and be expanded to assess other miniproteins with the goal to aid future experimental work.