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. 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. 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. 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>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>

Voltage-gated K+ (Kv) channels are known to cause serious disease upon their malfunction. Kv channels desensitised to voltage show inability to fully repolarise the membrane in excitable cells, which can make the membrane hyperexcited and in turn cause seizures such as in epilepsy, periodic ataxia or heart arrhythmia. Therefore, enhancers of Kv channels could serve as potential drugs. Some of these enhancers are polyunsaturated fatty acids and resin-acids which bind at the proteinlipid surface and affect the movement of the voltage sensor in the channel by a mechanism called the lipoelectric effect. To explore the lipoelectric modulation mechanism, we have performed an extensive computational study including docking and molecular dynamics simulations on resin-acid derivatives added to a model potassium channel called Shaker. Four derivatives, Wu32 and Wu50 that excite the channel and thus induce repolarisation of the membrane, as well as Wu18 and Wu27, who were found to be non-potent in previous experimental studies, have helped to point out a novel binding site in Shaker. The site is located between the pore and voltage-sensing domain of the channel and is in direct contact with the first gating charge arginine, R1, and the residue W454. We hypothesize that it is possible for resinacid derivatives to directly bind to the voltage-sensor when it is in an activated state, prolonging the time Shaker stays open. Further experimental studies on Shaker and human homologs are now needed to test our hypothesis. Therefore, we suggest recording the sensitivity of Shaker towards potent derivatives in combination with mutations of W454. If our findings of the novel binding site are correct, the suitability of Shaker as a model system for human Kv channel modulation by lipoelectric modulators can be questioned as W454 is replaced by small hydrophobic side chains in mammalian Shaker homologs.

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. / 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. / <p>QC 20171003</p>

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. / <p>QC 20171117</p>

Living cells are highly complex, making it an extremely challenging task to understand how they function. A possible solution is the bottom-up assembly of non-living components and building up life-like features from scratch, i.e., using synthetic cells as a tool to understand the basic characteristics of life. One such chassis for synthetic cells are liposomes, which, like the cell membrane of living cells, are made of phospholipids. As living cells grow, lipids are incorporated into their membrane in order to cope up with the volume increase of the cell. In a similar fashion, a variety of ways are currently being investigated to achieve growth of synthetic cells. Few examples include incorporation of fatty acids from the surrounding environment, reconstituting the enzymes for fatty acid or lipid biosynthesis in the liposome, or by carrying out the synthesis of artificial membrane components through the external addition of precursor molecules. Here, we demonstrate the membrane-tension mediated growth of giant unilamellar vesicles (GUVs) by fusing sub-micrometre-sized feeder vesicles to them. We use a recently developed microfluidic technique, octanol-assisted liposome assembly (OLA), to produce cell-sized (~10 μm) GUVs on-chip. Following the density-based separation of the liposomes from the waste product (1-octanol droplets), we supply small unilamellar vesicles (SUVs, ~30 nm in diameter) which act as a lipid reserve for growth by fusing with the GUVs. The lipids molecules, being very stable in bilayer conformation, require energy to reorient themselves and undergo membrane fusion. We show that increased membrane tension of GUVs can act as a sole driver to carry out multiple fusion events and cause significant growth. By placing a mass population (&gt;1000) of GUVs in a sufficiently hypotonic solution (delta c 3−5 mM), we build up the membrane tension (~10 mN/m) driving multiple SUV-GUV fusionevents, eventually doubling the volume of a part of the population. We probe a variety of lipid compositions, including hybrid (composed of lipids and fatty acids) GUVs and find the growth to be dependent on the lipid composition. Maximum growth is obtained when using a hybrid system, as compared to pure lipids. Our results show the possibility to use a protein-freeminimal system to induce growth in a minimalistic manner and the demonstrated highthroughput microfluidic approach may have useful implications towards realizing an autonomous entity capable of undergoing a continuous growth-division cycle.

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.

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. / 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.

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. / 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.

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. / <p>QC 20170523</p>