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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>

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

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

The intracellular environment in which most proteins fold and function contains a range of biomolecules that results in significant volume exclusion, thus contrasting to the dilute buffer conditions common to most in vitro studies. In addition to intracellular macromolecular crowding, cells are ionic in nature, and although the Hofmeister series of ions has its origin in a work from 1888, much is still unclear concerning how small, charged ions affect protein properties. This thesis summarizes in vitro work assessing the effects of macromolecular crowding and small ions on the biophysical properties of a model protein -- Desulfovibrio desulfuricans flavodoxin. Flavodoxin is a small (15.7 kDa), single domain, cytoplasmic protein with alpha-helical and parallel beta-sheet secondary structural elements arranged in one of the five most common protein folds (the flavodoxin-like fold). Using a range of biophysical/spectroscopic methods (e.g., circular dichroism (CD), fluorescence, calorimetry, stopped-flow mixing) along with synthetic crowding agents (e.g., Ficoll and dextran), I have found that macromolecular crowding increases the secondary structural content of folded flavodoxin (toward that found in the crystal structure), increases flavodoxin thermal stability, and affects both the accumulation of a misfolded intermediate and the rate of proper protein folding. Collaborative in silico simulations employing Go-like modeling of apoflavodoxin in the presence of large, inert crowding agents agrees with my in vitro work and provides structural and mechanistic information with residue-specific resolution. We also found that small cations and anions in physiologically relevant concentrations (&le; 250 mM) increase flavodoxin thermal stability significantly. Both cations and anions in higher concentrations (300 mM-.75 M) affect oppositely charged proteins similarly suggesting that surface electrostatic charge plays only a minor role in mediating ionic effects on protein thermal stability. At all ion concentrations, ionic effects on protein stability are correlated to ion hydration (and thus the Hofmeister series). Our work suggests a dominant role for the peptide bond in coordinating ions at higher concentrations. This thesis work suggests that the crowded and ionic nature of the intracellular milieu can elicit changes to the structure, dynamics, stability, and folding mechanism of proteins which may not be captured in vitro using dilute buffer conditions.

The suddenly global outbreak of 2009 H1N1 Flu reminds human being the danger and severity of influenza virus. The gene of the new strain come from five different influenza viruses: North American swine influenza, North American avian influenza, human influenza, and two swine influenza viruses typically found in Eurasia. The recombination of gene in virus evolvement makes it more and more important to understand the evolutionary characteristics of influenza. Hemagglutinin (HA), embedded on the surface of influenza virus, is one of two virally-coded integral envelope proteins of the virus. The three primary functions of hemagglutinin (HA) include receptor binding, membrane fusion, and antigenic variation. Study of HA structure and its evolutionary mechanism is crucial to fully understand influenza virus. In the first chapter, a study of diversifying selective pressure on influenze B virus hemagglutinin was reported. All the positively selected sites were located in the four epitopes (120-loop, 150-loop, 160-loop and 190-helix) of HA, and all of them have been identified in previous studies. This supports a predominant role of antibody selection in HA evolution. In the second chapter, we studied positive selection analysis of 2009 HINT Flu. Among a subgroup of human A(H1N1) HAs between 1918&sim;2008, we found strong diversifying (positive) selection at HAI 156 and 190. We also analyzed the evolutionary trends at HAI 190 and 225 that are critical determinants for receptor-binding specificity of A(H1N1) HA. Additional analysis of directional selection was also employed for H1N1 gene data.

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

<p>Embryonic development involves a precisely orchestrated interplay between gene expression, tissue movement and cell-shape changes. Using time-lapsed <italic>in vivo</italic> confocal microscopy we investigate the time-dependence of cell-shape changes for essentially all of the cells in the amnioserosa tissue during the early-to-late stages of dorsal closure in <italic>Drosophila melanogaster</italic>. Dorsal closure is a critical stage during embryogenesis, where two epidermal tissues are brought together by the force producing machinery in the surrounding tissues, including the amnioserosa tissue. The environment these cells exist in is dominated by viscous forces, making the observable kinematics the result of active contractile force imbalances along the cell peripheries. Our image contrast is due to GFP-DE-cadherin, a relatively bright fluorescent construct that localizes at cell-cell junctions. Using custom written segmentation software we quantify cell apical areas from confocal images. By considering the kinematics of individual cells we investigate the forces produced by the amnioserosa tissue. We confirm previous observations of area pulsations or oscillations and that, within the dorsal opening, areas of peripheral amnioserosa cells are smaller than the areas of interior cells [Fernandez et al., 2007, Gorfinkiel et al., 2009, Solon et al., 2009, Blanchard et al., 2010]. In addition to oscillations, we find that cells in the low-Reynolds environment of the amnioserosa tissue exhibit ingression processes, a persistent loss of apical area resulting in the internalization of the cell. We develop an empirical model that quantifies the kinematics of the ingression processes of a substantial fraction of the amnioserosa cells. We also account for these observations with a biophysical model that quantifies the (spatially averaged) net force from experimental data and explicitly treats the dynamics of oscillations and ingression. Utilizing both models, we find that approximately half of the amnioserosa cells exhibit a loss of apical cross-sectional area dominated by an irreversible ingression process. For these cells, a transition is resolved from largely reversible oscillations to the onset of an ingression process. We also investigate variability in cell kinematics according to location within the dorsal opening and we find that cells ingress along each leading edge, in addition to previously observed ingression associated with the zipping process or associated with apoptosis. We attribute cell-to-cell variability in the maximum rate of constriction during the ingression processes to be a consequence of variability in the magnitude of force produced by the cytoskeleton. Finally, we investigate invariant properties, i.e., time-independent, global properties of dorsal closure and find nearly constant rates of completion of ingression processes as well as a constant of proportionality that relates the area of the dorsal opening to its two principle axes.</p> / Dissertation

EcoRI endonuclease is a very useful tool to study the structural mechanism of protein-DNA recognition. In this work, wild type EcoRI Endonuclease and EcoRI Endonuclease mutant RS187 were purified to high purity. Crystals of wild type EcoRI Endonuclease-DNA 13mer complex have been obtained with good size and shape. Some small crystals of EcoRI Endonuclease RS187-DNA 13mer complex were also grown.

Methyl-CpG binding protein 2 (MeCP2) is a protein associated with transcriptional repression of other proteins and its various mutants are found in Rett syndrome patients, which is a severe neurodevelopmental disease found in 1/15,000 females. It has specific binding affinity to methyl-CpG domain but also bind to architectural DNA structure. Here, its binding dynamics to Holliday junction structure was investigated by using single molecular detections; scanning confocal fluorescence microscope and wide-field evanescent field fluorescence microscope. Through these studies, we could distinguish the difference of transitions of the substrate with and without MeCP2. The effect of this MeCP2 binding to 4WJ will be discussed in terms of the structures and transition times.