The diploma thesis deals with the registration of images taken with the multimodal holographic microscope (MHM). The summary covers the fluorescent and holographic microscopy, and the multimodal holographic microscope combining both these microscopy types. Every pair of the images needs to be aligned in order to gain new information by combining both image types. The thesis contains an algorithm that registers images by phase correlation as well as a process created in MATLAB in accordance with the algorithm. The most important procedure parameters’ influence on the registration success is described and the results are annotated.

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance – PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol – using 2H solidstate nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.

The Gram-negative bacterium Pseudomonas aeruginosa is a life-threatening nosocomial pathogen due to its generally low susceptibility toward antibiotics. Furthermore, many strains have acquired resistance mechanisms requiring new antimicrobials with novel mechanisms to enhance treatment options. Proline-rich antimicrobial peptides, such as the apidaecin analog Api137, are highly efficient against various Enterobacteriaceae infections in mice, but less active against P. aeruginosa in vitro. Here, we extended our recent work by optimizing lead peptides Api755 (gu-OIORPVYOPRPRPPHPRL-OH; gu = N,N,N′,N′-tetramethylguanidino, O = L-ornithine) and Api760 (gu-OWORPVYOPRPRPPHPRL-OH) by incorporation of Ile-Orn- and Trp-Orn-motifs, respectively. Api795 (gu-O(IO)2RPVYOPRPRPPHPRL-OH) and Api794 (gu-O(WO)3RPVYOPRPRPPHPRL-OH) were highly active against P. aeruginosa with minimal inhibitory concentrations of 8–16 and 8–32 μg/mL against Escherichia coli and Klebsiella pneumoniae. Assessed using a quartz crystal microbalance, these peptides inserted into a membrane layer and the surface activity increased gradually from Api137, over Api795, to Api794. This mode of action was confirmed by transmission electron microscopy indicating some membrane damage only at the high peptide concentrations. Api794 and Api795 were highly stable against serum proteases (half-life times >5 h) and non-hemolytic to human erythrocytes at peptide concentrations of 0.6 g/L. At this concentration, Api795 reduced the cell viability of HeLa cells only slightly, whereas the IC50 of Api794 was 0.23 ± 0.09 g/L. Confocal fluorescence microscopy revealed no colocalization of 5(6)-carboxyfluorescein-labeled Api794 or Api795 with the mitochondria, excluding interactions with the mitochondrial membrane. Interestingly, Api795 was localized in endosomes, whereas Api794 was present in endosomes and the cytosol. This was verified using flow cytometry showing a 50% higher uptake of Api794 in HeLa cells compared with Api795. The uptake was reduced for both peptides by 50 and 80%, respectively, after inhibiting endocytotic uptake with dynasore. In summary, Api794 and Api795 were highly active against P. aeruginosa in vitro. Both peptides passed across the bacterial membrane efficiently, most likely then disturbing the ribosome assembly, and resulting in further intracellular damage. Api795 with its IOIO-motif, which was particularly active and only slightly toxic in vitro, appears to represent a promising third generation lead compound for the development of novel antibiotics against P. aeruginosa.

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Indiana University-Purdue University Indianapolis (IUPUI) / Cancer is still among those diseases that prominently contribute to the numerous deaths that are caused each year. But as technology and research is reaching new zeniths in the present times, cure or early detection of cancer is possible. The detection of rare cells can help understand the origin of many diseases. The current study deals with one such technology that is used for the capture or effective separation of these rare cells called Lab-on-a-chip microchip technology. The isolation and capture of rare cells is a problem uniquely suited to microfluidic devices, in which geometries on the cellular length scale can be engineered and a wide range of chemical functionalizations can be implemented. The performance of such devices is primarily affected by the chemical interaction between the cell and the capture surface and the mechanics of cell-surface collision and adhesion. This study focuses on the fundamental adhesion and transport mechanisms in rare cell-capture microdevices, and explores modern device design strategies in a transport context. The biorheology and engineering parameters of cell adhesion are defined; chip geometries are reviewed. Transport at the microscale, cell-wall interactions that result in cell motion across streamlines, is discussed. We have concentrated majorly on the fluid dynamics design of the chip. A simplified description of the device would be to say that the chip is at micro scale. There are posts arranged on the chip such that the arrangement will lead to a higher capture of rare cells. Blood consisting of rare cells will be passed through the chip and the posts will pose as an obstruction so that the interception and capture efficiency of the rare cells increases. The captured cells can be observed by fluorescence microscopy. As compared to previous studies of using solid microposts, we will be incorporating a new concept of cylindrical shell micropost. This type of micropost consists of a solid inner core and the annulus area is covered with a forest of silicon nanopillars. Utilization of such a design helps in increasing the interception and capture efficiency and reducing the hydrodynamic resistance between the cells and the posts. Computational analysis is done for different designs of the posts. Drag on the microposts due to fluid flow has a great significance on the capture efficiency of the chip. Also, the arrangement of the posts is important to contributing to the increase in the interception efficiency. The effects of these parameters on the efficiency in junction with other factors have been studied and quantified. The study is concluded by discussing design strategies with a focus on leveraging the underlying transport phenomena to maximize device performance.

10 SUMMARY Proteins represent one of the basic building blocks of all organisms. To understand their function at the molecular level is one the critical goals of current biological, biochemical and biophysical research. It is important to characterise all aspects that affect the localisation of proteins into different compartments with specific functions, the dynamic structure of proteins and their role in multiprotein assemblies, because altering these properties can lead to various diseases. Most of the proteomic studies are nowadays performed using biochemical approaches that allow us to study multicellular organism or tissue at once. The disadvantage of these methods is complex preparation of sample and the need for a large number of cells, which leads to the loss of information at the molecular level and in individual cells. On the contrary, microscopy can provide rather detailed information about proteins of interest and at the level of a single cell. A variety of fluorescence microscopy methods in combination with recombinant DNA techniques were applied to elucidate subcellular localisation of transmembrane adaptor proteins (TRAPs) in human lymphocytes and their nanoscopic organisation at the plasma membrane. Linker of activation of T lymphocytes (LAT), phosphoprotein associated with...

Synuclein, Parkinson's disease, network activity, neuron, tau protein, neurodegeneration, connectivity, cAMP,

Natrium-kaliumpumpen spelar en väsentlig roll i en rad fysiologiska funktioner då den upprätthåller den elektrokemiska gradienten över cellmembranet. Ytterligare så är störningar i dess funktion associerade med flera neurologiska sjukdomar. Proteinet är en heterodimer av α– och β–subenheter, ibland även associerat med en tredje γ (FXYD) subenhet, vilket gör det problematiskt att studera dess högre ordningens organisation i cellmembranet med hjälp av konventionella, relativt storskaliga inmärkiningsprober såsom antikroppar. Inkorporering av icke-kanoniska aminosyror är ett nyutvecklat och växande område som erbjuder en lösning. Genom CuAAC– och SPIEDAC–klickkonjugationsreaktioner kan organiska färgämnen (fluoroforer) snabbt och specifikt fästas i sidokedjor med motsvarande reaktiva grupper på jonpumpen, vilket skapar en liten och icke-invasiv inmärkningsprob för fluorescensmikroskopi. För att specifikt studera alla tre subenheter samtidigt krävs inmärkning med tre olika fluoroforer). Syftet med detta projekt var att lyckas med trefärgsinmärkning genom inkorporering av icke-kanoniska aminosyror, och därigenom underlätta studerandet av hur natrium-kaliumpumpens subenheter ordnar sig i cellmembranet. Transient transfekterade HEK293T-celler med membraninmärkta jonpumpar studerades med hjälp av fluorescensmikroskopi, vilket kompletterades med gelfluorescensavbildning och immunoblotting. Samtidigt gjordes proteinuttryck och tvåfärgsinmärkning av alla nonsenskodonmuterade subenheter i kombination med varandra och var synlig i proteingel, där endast α och β tidigare hade samuttryckts. α/γ parinmärkning visade sig framgångsrik när de samtransfekterades med β av vildtyp. En autofluorescenseffekt i en av färgkanalerna påverkade resultaten för mikroskopin. Trefärgsinmärkning observerades inte i gelen, och uttrycket av subenheterna (varav α var ersatt för detta experiment) var i stort sett obefintligt. Otydlighet består därmed huruvida trefärgsinmärkning eller trippelsamuttryck är möjligt med de bioortogonala translationssystemen som användes i detta projekt på jonpumpen. / Na+/K+-ATPase is an essential ion pump protein in a host of physiological functions as it maintains the electrochemical gradient across cell membranes. Additionally, its dysfunction is implicated in several neurological diseases. The protein is a heterodimer of α and β subunits, occasionally associated with a third γ (FXYD) subunit, which makes studying its higher order organization in the cell membrane difficult using conventional, relatively large scale labeling probes such as antibodies. Non-canonical amino acid incorporation is an emerging field which offers a solution. Via CuAAC and SPIEDAC click conjugation reactions, organic fluorophores can be specifically attached to the side chains of residues of the ion pump with corresponding reactive moieties, creating a small and noninvasive probe for fluorescence microscopy imaging. In order to specifically image all three subunits concurrently, three color labeling is required. The objective of this project was to achieve three color labeling via non-canonical amino acid incorporation to aid in the study of the cell membrane localization of the subunits of Na+/K+-ATPase. Fluorescence microscopy of transiently transfected and live cell labeled HEK293T cells was complemented by in gel fluorescence imaging and immunoblotting. Coexpression and two color labeling of all nonsense codon subunit mutants in combination was shown in gel, of which only α and β had previously been coexpressed. α/γ dual labeling proved successful when cotransfected with wild type β. An autofluorescent effect in one of the color channels compromised the microscopy results. Three color labeling was not observed in gel, and expression of the subunits (including a substitute for α) was middling to absent. It remains unclear whether three color labeling or triple coexpression is a possibility with the bioorthogonal translation systems used in this project.

In biology, it is common to study cultured cells (in vitro) with fluorescence time-lapse microscopy. The cells are recorded for longer period of time and can later be viewed at an accelerated speed. During the acquisition some live cells tend to migrate. This can be a problem if the cell’s migration speed is high enough to move outside the field of view (FOV) during the acquisition time. The cells that moves outside the FOV can no longer be recorded and the information about them will be lost. This thesis presents scripts that have been developed for ZEN (blue) to be able to track a specific migrating cell of interest in real-time with automated control of imaging parameters. The microscope stage position is modified on-the-fly to have the tracked cell in the center of the FOV for the whole experiment. Three different types of experiments to track migrating NK cells were performed with the scripts. The results show that the scripts were able to track one NK cell for more than 1 hour in both conventional wide-field and lattice light-sheet microscopy. The segmentation was inaccurate when one or more objects were in close proximity to the tracked cell. By applying a watershed algorithm the segmentation result can be improved in some cases. / Inom cellulär biologi är det vanligt att studera odlade celler (in vitro) med time- lapse-mikroskopi. Flertals bilder tas på cellerna under en längre tidsperiod och när experimentet är klart så kan man titta på bilderna som en video. Under förvärvet av bilderna så tenderar vissa levande celler att migrera. Ett problem som kan uppstå är om cellens migrationshastighet är tillräckligt hög för att röra sig utanför synfältet under anskaffningstiden. De celler som rör sig utanför synfältet kan inte längre avbildas och informationen om dem kommer att gå förlorad. I denna avhandling presenteras programmeringskoder som har utvecklats för ZEN (blue) som kan spåra en specifik migrerande cell i realtid med automatiserad kontroll av bildbehandlings parametrar. Mikroskopets scenposition modifieras under experimentets gång för att få den spårade cellen kontinuerligt i mitten av synfältet. Tre olika sorters experiment i kombination med programmeringskoderna utfördes för att spåra NK-celler. Resultaten visar att programmeringskoderna lyckades spåra en NK-cell i mer än 1 timme i både ett bredfältsfluorescensmikroskop och ett lattice light-sheet mikroskop. Segmenteringen var felaktig när ett eller flera objekt var i närheten av den spårade cellen. Genom att tillämpa en watershed algoritm kan segmenteringsresultatet förbättras i vissa fall.