The coiled-coil motif is present in proteins from all kingdoms of life. Its structure is based on a repeating sequence of 7 amino acids with hydrophobic residues at positions 1 and 4, which folds into an alpha-helix. Two, or more, alpha-helices wind around each other based on hydrophobic interactions forming the coiled-coil. Structural variations include length, deviations from the canonical form based on the heptad repeat, as well as the orientation and number of alpha-helices. They are involved in a wide variety of cellular processes including vesicle tethering and signal transmission along their length. In order to transmit signal, the protein must be able to dynamically rearrange its structure. An outstanding example of a coiled-coil that needs to rearrange its structure to perform its function is the early endosomal tether EEA1, which has been shown to increase its flexibility upon binding to the active form of the small GTPase Rab5. That conformational change generates an entropic collapse that brings the ends of the protein closer to each other. Nevertheless, the recycling from the more flexible state to its original extended conformation was not addressed. Herein, the entropic collapse mechanism was further studied and the full EEA1 cycle between extended and flexible states described. In addition to these studies, other coiled-coil proteins were assessed to determine if they also experience a binding-induced entropic collapse. One of the strategies to investigate the entropic collapse mechanism was to compare the adhesive forces along the two alpha-helices of the EEA1 dimer in its extended and flexible conformations. To this end, an experiment was designed to unwind the dimer using optical tweezers, a force-spectroscopy method that uses a highly focused laser beam to manipulate microscopic objects. Each EEA1 monomer was attached to a distinct DNA piece using a site-specific enzymatic reaction. The DNA pieces were linked to two optically trapped micron-sized beads. And the distance between the optical traps increased to unwind the EEA1. A second strategy to investigate the entropic collapse was to evaluate EEA1 dynamics in solution using dual color fluorescence cross-correlation spectroscopy (dcFCCS). EEA1 C-termini was labeled with two different fluorophores. Fluctuations on fluorescent intensities caused by the dyes crossing a confocal volume were recorded over time. Based on an analysis of these fluctuations, a conformational change in EEA1 from semi-flexible to flexible upon addition of active Rab5 was described. This is in agreement with the previously reported entropic collapse. More importantly, EEA1 was shown to cycle between semi-flexible and flexible states by adding Rab5:GTP and waiting for the GTP to hydrolyse. To determine whether other proteins experience a binding-induced entropic collapse, coiled-coil proteins that share structural and functional similarities with EEA1 were evaluated. Rotary shadowing EM images of the target protein alone and binding with its suspected allosteric effector were compared. It was found that ELKS, a coiled-coil protein involved in vesicle trafficking, undergoes an increase in flexibility upon binding with the active form of Rab6. Thus, hinting that the entropic collapse may indeed be a general mode of action for at least a sub-group of long coiled-coil proteins. Overall, the major contributions of this thesis are to describe the full entropic collapse cycle on EEA1 and to show a second example of a coiled-coil protein experiencing a binding induced flexibility increase.:List of Figures List of Tables List of Equations List of Abbreviations 1 Introduction 1.1 EEA1 as an endosomal tether 2 Materials and Methods 2.1 Materials 2.2 Methods 2.2.1 Sub-cloning 2.2.2 Protein expression and purification 2.2.3 Protein-protein binding assays 2.2.4 Electron microscopy 2.2.5 Analysis of electron microscopy 2.2.6 Generation of DNA handles for protein-DNA conjugates 2.2.7 Adding SortaseA recognition site to EEA1 2.2.8 Protein-DNA conjugation3 2.2.9 Sample preparation for optical tweezers 2.2.10 Dual color labeling of EEA1 2.2.11 Fluorescence cross-correlation spectroscopy 2.2.12 Generation of dsDNA for dcFCCS calibration 2.2.13 RabGTPase nucleotide loading 2.2.14 Liposome preparation 2.2.15 MCBs preparation 3 Unwinding EEA1 coiled-coil domain 3.1 Introduction 3.1.1 Optical tweezers for EEA1 unwinding 3.1.2 SortaseA-catalysed ligation 3.2 Aims 3.3 Results 3.3.1 Optimization of SortaseA-catalysed ligation 3.3.2 Formation of EEA1-DNA handle conjugate 3.3.3 EEA1 unwinding experiments 3.4 Discussion 4 EEA1 entropic collapse is recyclable 4.1 Introduction 4.1.1 Advantages of dcFCCS vs FCS 4.1.2 Requirements for dcFCCS measurements 4.1.3 dcFCCS for end polymer dynamics analysis 4.2 Aims 4.3 Results 4.3.1 System preparation and dcFCCS calibration 4.3.2 Labelling of EEA1 4.3.3 Comparing FCS vs dcFCCS 4.3.4 EEA1 entropic collapse shown by dcFCCS 4.3.5 EEA1 flexibility change is recyclable 4.4 Discussion 5 Entropic collapse as a general mechanism 5.1 Introduction 5.2 Aims 5.3 Results 5.3.1 ELKS increases its flexibility upon binding active Rab6 5.3.2 p115-GM130 complex observed by rotary shadowing EM 5.4 Discussion 6 Conclusions and outlook References

The dissertation presents theoretical and experimental studies on the physical origin of the signal in photothermal microscopy of single particles. This noninvasive optical far field microscopy scheme allows the imaging and detection of single absorbing nanoparticles. Based on a heat-induced pertur- bation in the refractive index in the embedding medium of the nanoscopic absorber, a corresponding probe beam modification is measured and quantified. The method is well established and has been applied since its first demonstration in 2002 to the imaging and characterization of various absorbing particle species, such as quantum dots, single molecules and nanoparticles of different shapes. The extensive theoretical developments presented in this thesis provide the first quantitative assess- ment of the signal and at the same time enlarge its phenomenology and thereby its potential. On the basis of several approximation schemes to the Maxwell equations, which fundamentally gov- ern the interaction of light with inhomogeneities, several complementing models are devised which describe the photothermal signal both qualitatively and quantitatively. In succession an interdepen- dent and self-consistent set of theoretical descriptions is given and allows important experimental consequences to be drawn. In consequence, the photothermal signal is shown to correspond to the action of a nanoscopic (thermal) lens, represented by the spherically symmetric refractive index pro- file n(r) which accompanies the thermal expansion of the absorber’s environment. The achieved quantification allows the direct measurement of absorption cross-sections of nanoparticles. Further, a qualitatively new phenomenology of the signal is unraveled and experimentally demonstrated. The separate roles of the probing and the heating beams in photothermal microscopy is dismantled and the influence of their relative alignment shown to allow for a controlled adjustment of the effective detection volume. For the first time, both positive and negative signals are demonstrated to occur and to be the characteristic signature of the lens-like action on the probe beam. The detection of the probe beam’s modification is also shown to sensitively depend on the aperture used in the detection chan- nel, and a signal optimization is shown to be feasible. Also, a generalization of the detectable signal via the use of a quadrant photodiode is achieved. Specifically, measuring the far field beam deflec- tion the result of the beam passing the lens off-center manifests in a laterally split detection volume. Hereby, finally each classical photothermal spectroscopic techniques has been shown to possess its microscopic counterpart. Central to the understanding of this generalized and new phenomenology is a scalar wave-optical model which draws an analogy between the scattering of a massive particle wave-packet by a Coulomb potential and the deflection of a focused beam by a photonic potential connected with the thermal lens. The significance of the findings is demonstrated by its methodological implications on photother- mal correlation spectroscopy in which the diffusion dynamics of absorbing colloidal particles can be studied. The unique split focal detection volumes are shown to allow the sensitive measurement of a deterministic velocity field. Finally, the method is supplemented by a newly introduced sta- tistical analysis method which is capable of characterizing samples containing a heterogeneous size distribution.:Contents Bibliographic description Abbreviations 1 Introduction 2 Theoretical Background 2.1 The current literature on the subject of the photothermal signal 2.2 Thermal conduction, and the temperature field around heated nanoparticles 2.3 The linear thermo-refractive response and the thermal lens 2.4 MAXWELL equations and approximation schemes 2.4.1 The MAXWELL equations 2.4.2 HELMHOLTZ equations 2.4.3 Paraxial HELMHOLTZ equation for the field components 2.4.4 Geometrical optics and the eikonal ansatz 2.5 Diffraction and the optical resolution limit in far field microscopy 2.5.1 Transmission scanning microscopy 2.5.2 Point spread functions and aberrations 2.5.3 Scalar diffraction approximation for weakly focused beams 2.5.4 Vectorial diffraction for highly focused electromagnetic fields 2.5.5 Theoretical description of transmission signals 2.6 Elastic scattering of light 2.6.1 Overview of optical elastic scattering theory 2.6.2 The integral equation of potential scattering and the BORN approximation 2.6.3 The generalized LORENZ-MIE theory 2.6.4 The electromagnetic fields 2.6.5 Description of the incident field: beam shape coefficients 2.6.6 Multilayered scatterers 2.6.7 POYNTING vector and field decomposition 2.6.8 Energy balance & total cross-sections 2.6.9 Optical theorem & the extinction paradox 2.6.10 Small particle scattering: the RAYLEIGH-limit 2.7 Optical properties of gold nanoparticles & Surface plasmon resonances 2.7.1 Dielectric function of gold 2.7.2 Total cross-sections of plasmonic nanoparticles properties of gold nanoparticles & Surface plasmon resonances 2.8 (Hot) BROWNian motion, diffusion and their statistical analysis 2.8.1 (Hot) BROWNian motion 2.8.2 Diffusion and correlation analysis 2.8.3 Methods regarding the signal statistics of diffusing tracer particles 2.9 RUTHERFORD scattering of charged particles 2.9.1 Classical RUTHERFORD scattering 2.9.2 Quantum mechanical COULOMB scattering 3 Experimental Setup 3.1 Sample preparation 3.2 Photothermal microscopy setup 4 Photothermal Imaging: Results and Discussion 4.1 MAXWELL equations: Exact treatment of the PT signal 4.1.1 Angularly resolved powers: Fractional cross-sections 4.1.2 Incident power and background normalization 4.1.3 Fractional scattering and extinction cross-sections (off-axis) 4.1.4 Fractional scattering and extinction cross-sections (on-axis) 4.1.5 Small particle approximation(on-axis) 4.1.6 General properties of transmission scans 4.1.7 The thermal lens n(r) in the MIE-scattering framework 4.1.8 The photothermal signal F in the MIE scattering framework 4.2 Geometrical optics: Photonic RUTHERFORD scattering (ray optics) 4.2.1 FERMAT’s principle for a thermal lens medium 4.2.2 Gaussian beam transformation by a thermal lens 4.2.3 Experiments using weakly focused, i.e. nearly Gaussian beams 4.3 HELMHOLTZ equation: Photonic RUTHERFORD scattering (wave optics) 4.3.1 Plane-wave scattering 4.3.2 Focused beam scattering 4.3.3 Connection to the far field 4.3.4 Photothermal Rutherford scattering microscopy 4.3.5 Photothermal half-aperture measurements 4.4 Paraxial HELMHOLTZ equation: FRESNEL diffraction by a thermal lens 4.4.1 The diffraction integral and the phase mask for a thermal lens 4.4.2 The photothermal signal expressed via the image plane field 4.4.3 Experimental demonstration of the signal inversion 4.4.4 Connection to photothermal RUTHERFORD scattering 4.5 Plane-wave extinction & scattering by a thermal lens 4.5.1 The BORN approximation for the ideal and time-dependent thermal lens 4.5.2 The eikonal approximation for the ideal thermal lens and x>>1 4.5.3 Lessons to be learned from plane-wave scattering by thermal lenses 4.6 What is a lens? And is n(r) a lens? 5 Methodological Applications of the Results 5.1 Generalized photothermal correlation spectroscopy (incl. twin-PhoCS) 5.2 Photothermal signal distribution analysis (PhoSDA) 6 Summary and Outlook 6.1 Summary of the results 6.2 Outlook 7 Appendix 7.1 Material parameters 7.2 Calculation parameters 7.3 Interactive simulation scripts (Processing) 7.4 Vectorial scattering in the BORN-approximation 7.5 Details regarding the scattering framework 7.5.1 Connection between Gmn,TE,TM of Ref.1 and gmn,TE,TM in the GLMT 7.5.2 Off-axis BSCs including aberration (single interface) 7.5.3 Details on the incidence power Pinc 7.5.4 Details on the incidence power Pinc for arbitrary beams 7.5.5 Explicit expressions for the spherical field components of Es,i and Hs,i 7.5.6 Note on the time-dependence and the corresponding sign-conventions in M 7.5.7 Recurrence relation for Pn and tn 7.5.8 Gaussian beam shape coefficients: Off-axis 7.5.9 Multilayered Scatterer 7.5.10 POYNTING-vector and energy flow fields 7.5.11 Convergence 7.5.12 Further evaluations in the GLMT framework 7.5.13 Diffraction model: Comparison of angular PT signal pattern to the GLMT 7.6 Details on geometrical optics models 7.6.1 Geometrical optics: Exact solution r(f) for |bx|<1 7.6.2 Correspondences in photonic and partile RUTHERFORD scattering 7.6.3 On the difference in the definition of optical energy 7.6.4 Ray-opticsphotothermalsignal 7.6.5 Thick lens raytracing and the equivalent lens shape for a given aberration 7.7 Thermal lens around a wire of radius R 7.8 Twin-PhoCS: Graphic illustration of the CCF integrand Curriculum Vitae Publications Declaration Acknowledgements List of Tables List of Figures Bibliography

No description available.

Dynamic light scattering (DLS) is a method to size submicron particles by measuring their thermal motion (diffusion) in suspensions and emulsions. However, the validity of the Stokes-Einstein equation that relates the diffusion coefficient and the particle size is limited to spherical particles and very low concentrations. Within this thesis, DLS is used for the characterization of suspensions of pyrogenic silica which consists of fractal-like aggregates composed of sintered spherical primary particles. These structural features clearly complicate the understanding of DLS experiments and have been a severe obstacle to employing DLS as routine standard tool for the characterization of pyrogenic silica. The main objective of this thesis is therefore to evaluate the application of DLS in product development and quality assurance of pyrogenic silica industry, what essentially means to identify those structural properties of fractal aggregates which are measurable with DLS and to quantify the method’s sensitivity to changes in these properties. The investigations presented here are split up into four parts, simulations that establish a relation between structural and hydrodynamic properties, experiments validating the simulation results, the characterization of concentrated suspensions and the application-oriented analysis of DLS data for specific industrially relevant measurement tasks. / Die Dynamische Lichtstreuung (DLS) ist eine Messmethode zur Größenbestimmung submikroner Partikel. Dabei wird primär die stochastische Bewegung der Teilchen (Diffusion) in Suspensionen und Emulsionen bewertet. Die Stokes-Einstein Gleichung, die das Verhältnis zwischen gemessenem Diffusionskoeffizienten und Partikelgröße wiedergibt, ist jedoch nur für kugelförmige Teilchen, die in sehr niedriger Konzentration vorliegen, gültig. In der vorliegenden Arbeit wird die dynamische Lichtstreuung zur Charakterisierung von Suspensionen pyrogener Kieselsäure eingesetzt. Diese besteht aus fraktalen Aggregaten, die wiederum aus versinterten aber meist kugelförmigen Primärpartikeln zusammengesetzt sind. Diese strukturellen Eigenschaften erschweren die Anwendbarkeit der DLS bzw. die Interpretation der Messergebnisse und verhinderten bisher den Einsatz der DLS als Routinemethode zur Charakterisierung pyrogener Kieselsäuren. Das Hauptziel dieser Arbeit ist daher eine Bewertung der Möglichkeiten der DLS für die Produktentwicklung und Qualitätssicherung in der Herstellung pyrogener Kieselsäuren. Das bedeutet im Besonderen, dass sowohl die messbaren granulometrischen Eigenschaften als auch die Sensitivität der Methode bei Eigenschaftsänderungen ermittelt werden müssen. Die hier durchgeführten Arbeiten sind in vier Teile gegliedert: Simulationen, die eine Beziehung zwischen strukturellen und hydrodynamischen Eigenschaften herstellen, Experimente zur Validierung der Simulationsergebnisse, die Charakterisierung konzentrierter Suspensionen und die anwendungsorientierte Auswertung von DLS-Daten für spezifische industrierelevante Messaufgaben.

Endocytosis is one of the most fundamental mechanisms by which the cell communicates with its surrounding. Specific signals are transduced through the cell membrane by a complex interplay between proteins and lipids. Clathrin depended endocytosis is one of important signalling pathways which leads to budding of the plasmalemma and a formation of endosomes. The FCHo2 is an essential protein at the initial stage of the this process. In is a membrane binding protein containing BAR (BIN, Amphiphysin, Rvs) domain which is responsible for a membrane binding. Although numerous valuable work on BAR proteins was published recently, the mechanistic description of a BAR domain functionality is missing. In present work we applied in vitro systems in order to gain knowledge about molecular basis of the activity of the FCHo2 BAR domain. In our studies we used supported lipid bilayers (SLBs) and lipid monolayers as s model membrane system. The experiments were carried out with a minimal number of components including the purified FCHo2 BAR domain. Using SLBs we showed that the BAR domain can bind to entirely flat bilayers. We also demonstrated that these interactions depend on the negatively charged lipid species incorporated in the membrane. We designed an assay which allows to quantify the membrane tubulation. We found out that the interaction of the FCHo2 BAR domain with the lipid membrane is concentration dependent. We showed that an area of the bilayer deformed by the protein depends on the amount of the used BAR domain. In order to study the relation between the mobility of lipids and the activity of FCHo2 BAR domain we designed a small-volume monolayer trough. The design of this micro-chamber allows for the implementation of the light microscopy. We demonstrated that the measured lipid diffusion in the monolayer by our new approach is in agreement with literature data. We carried out fluorescence correlation spectroscopy (FCS) experiments at different density of lipids at the water-air interface.We showed that the FCHo2 BAR domain binding affinity is proportional to the mean molecular area (MMA). We additionally demonstrated that the increased protein binding is correlated with the higher lipid mobility in the monolayer. Additionally, by curing out high-speed atomic force microscopy (hsAFM) we acquired the structural information about FCHo2 BAR domains orientation at the membrane with a high spatio-temporal resolution. Obtained data indicate the BAR domains interact witheach other by many different contact sites what results in a variety of protein orientations in a protein assemble.

No description available.

A study was made in order to deduce whether certain chemical species, namely water (H2O) and carbon monoxide (CO), are present in the atmosphere of the exoplanet WASP-107 b, which lies about 200 light-years away from Earth in the constellation Virgo. The project was carried out at Uppsala University at the Department of Physics and Astronomy. This was done through the use of transmission spectroscopy and executed using a cross-correlation technique, one of the leading methods available today to extract exoplanetary atmospheric information. The data used was collected during a transit which occured in March 2022, originally gathered by the spectrograph CRIRES+ stationed at Very Large Telescope (VLT) in Paranal. WASP-107 b is a warm Jupiter-type planet, and since the aforementioned chemical species exhibit spectral lines mainly in the infrared (0.95-5.3 μm), it makes CRIRES+ a desirable instrument due to its specialization for working in the infrared. The data analysis was performed using several scripts built in Python with subsequent data-reduction methods. The data-reduction methods used for this purpose was the standard ESO CRIRES+ data reduction pipeline which includes removal of systemic sources of noise such as dead pixels and cosmic rays, and SysRem, which is an algorithm used to remove any trends with time and any constant features in time for each pixel time series. SysRem is currently one of the most efficient way available for doing so, and is commonly used in these types of studies. Several detection maps were then generated and studied in order to deduce whether a detection had been made or not. For this project, one exoplanet was examined and its atmosphere was probed for H2O and CO. The cross-correlation templates utilized were a combination of both species as well as one corresponding to only CO. The detection maps generated from the cross-correlation analysis initially suggested non-detections for all combinations of SysRem iterations and templates, except for two which presented features that might imply a detection but without any strong certainty. Those results indicate the possible existence of CO in the atmosphere of WASP-107 b, but further investigation is needed in order to determine their validity. / Denna rapport beskriver en studie som utförts för att undersöka ifall vissa kemiska arter, nämligen vatten (H2O) och kolmonoxid (CO), existerar i atmosfären kring exoplaneten WASP-107 b. Exoplaneten ligger cirka 200 ljusår bort från jorden i konstellationen Jungfrun. Arbetet utfördes på Uppsala universitet på institutionen för fysik och astronomi, eller Department of Physics and Astronomy. Detta gjordes huvudsakligen med hjälp av transmissionsspektroskopi och cross-correlation - en av de ledande metoderna idag för att analysera exoplanetära atmosfärer. Datan som använts för denna studie samlades in under en transit som skedde i mars 2022 med hjälp av spektrografen CRIRES+, stationerad vid Very Large Telescope (VLT) i Paranal. WASP-107 b klassas som en varm Jupiter, och eftersom de undersökta kemiska arterna huvudsakligen uppvisar spektrallinjer i det infraröda området (0.95-5.3 μm), är CRIRES+ ett sunt val då spektrografen är specialiserad på att undersöka infrarött ljus. Dataanalysen utfördes genom användningen av flertal script, byggda i Python med påföljande datareduktion. De datareduktionsmetoder som användes i detta syfte var ESO CRIRES+ standard data reduction pipeline, vilken inkluderar avlägsnandet av systematiska källor till brus såsom döda pixlar och den kosmiska bakgrundsstrålningen, och SysRem, vilket är en algoritm som används för att ta bort trender samt konstanta drag beroende på tid utmed varje pixelserie. I nuläget är SysRem en av de mer effektiva sätten att göra detta på, och är en vanlig metod i studier som denna. I detta projekt blev en exoplanet undersökt och dess atmosfär granskad för att se ifall H2O och CO förekommer i den. De cross-correlation templates som användes bestod av en som använde en kombination av båda kemiska arter, tillika en som endast detekterade CO. De detection maps som genererats från cross-correlation analysen föreslog först en ickedetektion för alla kombinationer av SysRem iterationer och templates, förutom två, vilka uppvisade signalement som möjligtvis indikerade en detektion, men utan särskild stark säkerhet. Dessa resultat föreslog en möjlig detektion av CO i atmosfären, men för att säkerställa detta krävs vidare undersökning.

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No description available.

The goal of this project is to study novel nanoscale excitation volumes, sensitive enoughto study individual chromophores and go on to study new and exciting self assemblyapproaches to this problem. Small excitation volumes may be engineered using light con-finement inside apertures in metal films. These apertures enhance fluorescence emissionrates, quantum yields, decrease fluorescence quenching, enable higher signal-to-noiseratios and allow higher concentration single chromophore fluorescence, to be studied byrestricting this excitation volume. Excitation volumes are reported on using the chro-mophore's fluorescence by utilising fluorescence correlation spectroscopy, which monitorsfluctuations in fluorescence intensity. From the correlation in time, we can find the res-idence time, the number of chromophores, the volume in which they are diffusing andtherefore the fluorescence emission efficiency. Fluorescence properties are a probe ofthe local environment, a particularly powerful tool due to the high brightness (quantumyield) fluorescent dyes and sensitive photo-detection equipment both of which are readilyavailable, (such as avalanche photodiodes and photomultiplier tubes). Novel materialscombining the properties of conducting and non-conducting materials at scales muchsmaller than the incident wavelength are known as meta-materials. These allow combi-nations of properties not usually possible in natural materials at optical frequencies. Theproperties reported so far include; negative refraction, negative phase velocity, fluorescenceemission enhancement, lensing and therefore light confinement has also been proposed tobe possible. Instead of expensive and slow lithography methods many of these materialsmay be fabricated with self assembly techniques, which are truly nanoscopic and otherwiseinaccessible with even the most sophisticated equipment. It was found that nanoscaled volumes from ZMW and HMMs based on NW arrays wereall inefficient at enhancing fluorescence. The primary cause was the reduced fluorescencelifetime reducing the fluorescence efficiency, which runs contrary to some commentatorsin the literature. NW based lensing was found to possible in the blue region of the opticalspectrum in a HMM, without the background fluorescence normally associated with a PAAtemplate. This was achieved using a pseudo-ordered array of relatively large nanowireswith a period just smaller than lambda / 2 which minimised losses. Nanowires in the traditionalregime lambda / 10 produced significant scattering and lead to diffraction, such that they werewholly unsuitable for an optical lensing application.