Introduction: Cancer vaccines are a therapeutic approach to initiate an antigen specific cytotoxic immune responses against tumors. Cancer vaccines are composed by an antigen (tumor peptide) and adjuvant. A peptide in combination with adjuvants effectively activate dendritic cells (DCs), the most efficient antigen presenting cells in our immune system. DCs prime and activate CD8+ cytotoxic T cells which generates an antigen specific response.Aim: Developing an assay to study the internalisation rout of an antigen-adjuvant conjugate in human dendritic cells by using live cell imaging. Method: Immobilisation of cells is necessary for the ability to perform live cell imaging for several hours. The immobilisation ability of three coatings, collagen type I, fibronectin and matrigel, at different concentrations were evaluated by using live cell imaging in a fluorescence microscope. The potential induction of activation of the cells were evaluated by using flow cytometry and ELISA. Results: Immature DCs internalise antigen-adjuvant conjugate more efficiently than mature and activated DCs. Therefore, it is important that the coating do not induce activation. Cells must also be immobilised for the possibility of long term detection. Collagen type I immobilised cells and induced activation in all investigated concentrations. Fibronectin and matrigel had concentration-dependent abilities to immobilise the cells. Matrigel did not activate the cells whilst fibronectin was concentration dependent. Conclusion: Matrigel immobilise the cells which enables long term single cell imaging without activation.

Control of cultivation conditions in the~live cell imaging extends the possibilities of biological experiments and makes the experimental results more reliable. In order to change the~cultivation conditions in a controlled manner and increase the reproducibility of the experiments, it is necessary to reduce the amount of manual operations and replace them with automated procedures. Therefore, the concept of a new automated culture device (bioreactor) was created. This device controls the exchange of medium in the observation chamber, ensures the circulation and exchange of the atmosphere and controls its composition. The bioreactor is intended for use in the Laboratory of Experimental Biophotonics. This laboratory is equipped with coherence-controlled holographic microscope (CCHM), which uses quantitative phase imaging (QPI) method. Thus, the bioreactor is adapted to the current requirements of this laboratory and optical elements of the bioreactor meet the requirements of the QPI method. This text specifies the cultivation conditions of the living cells and summarizes, how the conditions could be controlled in the live cell microscopy. Next some commercially available culture devices are described and assessed, whether they are convenient for the~use in Laboratory of Experimental Biophotonics. The crucial part of the thesis is the~design, construction and testing of the new bioreactor.

The goal of this diploma thesis was using of a coherence-controlled holographic microscope in cell’s life research. A brief history of interference microscopy and it’s applications in biology is described. Also other microscopy techniques routinely used for transparent objects imaging are mentioned and the biology of cell’s life cycle briefly explained. Characteristics describing the shape of a cell were proposed and tested with respect to identification of particular phases of its life cycle. The method of dynamic phase differences was modified in order to distinguish the internal motion of cell’s mass from the movement of the whole cell. Selected characteristics were used to evaluate observations carried out with the holographic microscope and the possibilities of their further applications were depicted. In conclusion, obtained findings were summarized and modifications of microscope construction as well as data-processing software were suggested.

Die Zellform ist wichtig für die Ausübung der Zellfunktion und spielt darüberhinaus eine essenzielle Rolle bei der Entwicklung eines Zellhaufens zu einem mehrzelligen Organismus. Dabei wird die Zellform neben biochemischen auch von biophysikalischen Prozessen beeinflusst: Zellkräfte sind ebenso beteiligt wie räumliche Einschränkung. Der Umfang der Wechselwirkung zwischen Umgebung, Zellform und Zellfunktion ist jedoch im Detail oft unverstanden. Ziel dieser Arbeit war daher, eine umfassende Charakterisierung von Zellen in räumlicher Einschränkung durchzuführen, um Aussagen zur Beeinflussung von Zellmorphologie und der Kraftentwicklung zu gewinnen. In dieser Arbeit wurde die Reaktion humaner Primärzellen (HUVECs) auf laterale Einschränkung untersucht. Die Zellen wurden dafür sowohl auf Glas- als auch auf Hydrogel-Substraten kultiviert, die mittels Mikrokontaktdruck von Fibronektin mit Streifenmustern im Breitenbereich von 5μm bis 80μm strukturiert worden waren. Die Zellen wurden nach der Phase der initialen Adhäsion (> 1 h) hinsichtlich ihrer allgemeinen Morphologie, des Erscheinungsbildes ihres Aktinskeletts und ihres Zellzugkraftverhaltens quantitativ beschrieben. Zusätzlich erfolgten Lebendzellmessungen, um die Dynamik des Aktinskeletts und der Zellzugkräfte zu charakterisieren. Die laterale Einschränkung führte zur strukturellen und funktionellen Adaption der Zellen. Da die Zelllänge nur geringfügig von der Streifenbreite abhing, kam es durch die seitliche Einschränkung zu einer Flächenabnahme bei gleichzeitiger Erhöhung des Zellseitenverhältnisses, wovon auch der Zellkern betroffen war. Die Ausrichtung der Aktinfasern korrelierte stark mit der Zellelongation und Zellen auf schmalen Streifen zeigten ein geringer vernetztes Aktinskelett. Messungen der Aktindynamik ergaben einen einwärts gerichteten Transport von Stressfasern. Weiterhin wurde eine Abnahme der Zugkräfte mit zunehmender Einschränkung gemessen, während gleichzeitig eine Polarisierung der Zugkräfte stattfand. Das beobachtete Verhalten der struktur- und funktionsbezogenen Zellparameter konnte gut durch die laterale Einschränkung erklärt werden, sodass die vorliegende Arbeit zu einem besseren Verständnis der Zellanpassung an räumliche Einschränkung beitragen konnte. / Proper cell shape is a precondition for the proper performance of specialized cells and changes of cell shape are paramount for the development from a cell cluster to an adult organism. Cell shape can be regulated biochemically and also biophysically, e. g., by involvement of cellular force generation and spatial confinement. However, the understanding of the interaction between exterior space, cellular form, and function is incomplete. Therefore, the aim of this thesis was to thoroughly characterize cells in spatial confinement in order to better understand how cell morphology and force generation can be linked. During the course of this work, the adaptation of human primary cells (HUVECs) to lateral constraints was investigated. Cells were seeded on both glass and hydrogel substrates which had been micropatterned with fibronectin by microcontact printing. The structures were composed of stripes with varying width (5–80 μm). After initial adhesion had taken place (> 1 h), cell morphology, actin cytoskeleton architecture, and cell traction forces were quantified. In addition, measurements were performed on live cells in order to better understand the dynamics of the actin cytoskeleton and the cell traction forces. Laterally confined cells showed both structural and functional changes. Because cell length was only weakly dependent on stripe width, cells in strong lateral confinement were highly elongated and had decreased spread areas, which also affected the nucleus. The orientation of actin fibers was strongly linked to cell elongation. In cells on narrow stripes, a reduced actin cytoskeleton was observed, i.e., with a lower degree of interconnectivity. Time resolved analysis revealed an inward transport of actin fibers. Furthermore, cell force generation was shown to be impaired on narrow stripes, most likely due to decreased cell spread area. At the same time, force polarization strongly increased in cells in strong lateral confinement. This study demonstrated how various cellular parameters, both linked to cell structure and function, are influenced by lateral confinement and by each other, thereby contributing to a better understanding of cell adaptation to spatial constraint.

Plant oriented movements, or tropisms allow the plant to actively respond to environmental stimuli to get more light, better access to nutrients and to grow roots deeper into the soil. Gravitropism drives the growth of roots along the gravity vector. Perception of gravity is triggered by the sedimentation of statoliths in columella root cap, but the exact signalling pathway behind this process is not known. Perception of gravity results in an unequal redistribution of the phytohormone auxin in the outer cell layers which leads to different rate of growth on the root's upper and lower side and bending of the root. The changes in auxin redistribution are accompanied by changes in apoplastic pH. Knowing an exact pattern of these pH changes could shed light on the mechanisms laying behind the gravitropic response pathway. While microelectrodes can be used to measure pH precisely, they are not suitable for the long-term imaging of growing roots. In the past few years, several pH sensitive dyes and genetically encoded sensors emerged. These can be used for long-term live in vivo imaging of pH changes in growing roots. In this thesis, I analysed the performance of several published pH sensitive genetically encoded sensors and available dyes in the roots of Arabidopsis thaliana. I observed that dyes varied...

Today new drugs are tested on cell cultures in wells to minimize time, cost, andanimal testing. The cells are studied using microscopy in different ways and fluorescentprobes are used to study finer details than the light microscopy can observe.This is an invasive method, so instead of molecular analysis, imaging can be used.In this project, phase-contrast microscopy images of cells together with fluorescentmicroscopy images were used. We use Machine Learning to predict the fluorescentimages from the light microscopy images using a strategy called In-Silico Labeling.A Convolutional Neural Network called U-Net was trained and showed good resultson two different datasets. Pixel-wise regression, pixel-wise classification, andimage classification with one cell in each image was tested. The image classificationwas the most difficult part due to difficulties assigning good quality labels tosingle cells. Pixel-wise regression showed the best result.

No description available.

Les cellules murales du cerveau sont un groupe de cellules neurovasculaires qui présentent une hétérogénéité moléculaire, morphologique et fonctionnelle exceptionnelle. Celles en contact avec les plus petis vaisseaux du cerveau, les péricytes du lit capillaire moyen sont connues pour être essentielles à l'homéostasie cérébrale, bien que leur capacité contractile ait longtemps été débattue. Cependant, nombre de leurs propriétés physiologiques, telles que leurs mécanismes de signalisation calcique, n'ont pas encore été élucidées. Cette thèse vise donc à identifier les mécanismes cellulaires de la signalisation calcique des péricytes des capillaires cérébraux. Dans le chapitre 2, nous utilisons la pharmacologie et l'imagerie des péricytes cérébraux exprimant l'indicateur de calcium GCaMP6f (provenant de souris transgéniques PDGFRβ-Cre::GCaMP6f) pour découvrir ces mécanismes. Contrairement aux péricytes engainants dont la signalisation du calcique dépend des canaux calcique voltage-dépendants, nous constatons que les signaux calcique des péricytes capillaire moyen sont indépendants des canaux calcique voltage-dépendants. Au contraire, nous constatons que les signaux calciques transitoires des pericytes du lit capillaire moyen sont inhibés par l'élimination du Ca2+ extracellulaire, l'inhibition des canaux Orai opérés par les réserves, le blocage du remplissage des réserves du réticulum endoplasmique, ainsi que l'inhibition des récepteurs de la ryanodine (RyRs) et des récepteurs de l'inositol trisphosphate (IP3Rs). Nous constatons également que l'entrée de Ca2+ opérée par les réserves peut être induite par la déplétion des réserves du réticulum endoplasmique et inhibée par les bloqueurs d'Orai dans les pericytes du lit capillaire moyen, et que l'influx basal de Ca2+ est largement dépendant de la déplétion des réserves. Enfin, nous montrons que l'entrée de Ca2+ opérée par les réserves d'Orai amplifie les élévations de Ca2+ cytosolique en réponse au vasoconstricteur endothéline-1. Nous concluons que la signalisation calcique dans les pericytes du lit capillaire moyen, qu'elle soit spontanée ou induite de façon agoniste, est régulée par le couplage entre la libération des réserves du réticulum endoplasmique et les voies d'influx opérées par les réserves. / Brain mural cells are a grouping of neurovascular cells that display exceptional molecular, morphological, and functional heterogeneity. Mid-capillary pericytes, the mural cells which contact the smallest vessels of the brain, are known to be critical to brain homeostasis, and their contractile ability has long been debated. However, many of their physiological properties, such as their Ca2+ signaling mechanisms, have not been elucidated. This thesis aims to uncover the cellular mechanisms of brain mid-capillary pericyte Ca2+ signaling. In chapter 2, we harness pharmacology and imaging of brain pericytes expressing the calcium indicator GCaMP6f (from transgenic PDGFRβ-Cre::GCaMP6f mice) to uncover these mechanisms. In contrast to ensheathing pericytes whose Ca2+ signaling is dependent on voltage-gated Ca2+ channels (VGCCs), we find that mid-capillary pericyte Ca2+ signals are independent of VGCCs. Instead, we find that mid-capillary pericyte Ca2+ transients are inhibited by removal of extracellular Ca2+, inhibition of store-operated Orai channels, blockade of endoplasmic reticulum store filling, as well as inhibition of ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs). We further find that store-operated Ca2+ entry can be induced by endoplasmic reticulum store depletion and inhibited by Orai blockers in mid-capillary pericytes, and that basal Ca2+ influx is largely dependent on store depletion. Finally, we show that Orai store-operated Ca2+ entry amplifies cytosolic Ca2+ elevations in response to the vasoconstrictor endothelin-1. We conclude that both spontaneous and Gq-coupled protein receptor agonist-induced Ca2+ signaling in mid-capillary pericytes is regulated by coupling between endoplasmic reticulum store release and store-operated influx pathways.

In dieser Arbeit wurden die von Seitz et al. entwickelten forced intercalation (FIT)-Sonden zur mRNA-Charakterisierung in lebenden Zellen eingesetzt. Es erfolgte die Synthese verbesserter FIT-Sonden für die systematische Untersuchung der Aufnahme durch lebende Flp-In™ 293 T-REx™-Zellen. Dafür wurden sowohl hergestellte FIT-Sonden-Konjugate/Aggregate als auch kommerziell erhältliche Reagenzien, wie z.B. die Palmitinsäure und das porenbildende Enzym Streptolysin-O auf ihre Effizienz untersucht. Die optimalen Bedingungen für das Einbringen von DNA- und PNA-FIT-Sonden in Flp-In™ 293 T-REx™-Zellen lieferte das Enzym Streptolysin-O. Durch den simultanen Einsatz von drei unterschiedlichen Sonden (BO-, TO- und CB-markiert), komplementär zu drei verschiedenen Zielsequenzen, gelang es erstmals eine Dreifarben-Lebendzell-Bildgebung mit FIT-Sonden durchzuführen. Des Weiteren wurden TO-FIT-Sonden zur Unterscheidung verschiedener T-Zelllinien eingesetzt. Mithilfe eines kompetitiven Hybridisierungsexperiments konnte die spezifische Fluoreszenzemission der Sonden in den Zellen belegt werden. Untersuchungen mit zwei T-Zelllinien zeigten, dass TO-FIT-Sonden sowie terminal Cy7-markierte TO-FIT-Sonden eine erhöhte TO-Emission bei Vorhandensein der komplementären TCR-mRNA-Zielsequenz in den Zellen aufwiesen. Der terminale Cy7-Farbstoff bot mit einem zweiten Detektionskanal die Möglichkeit die Cy7-Intensität und die vorhandene TO-Intensität ins Verhältnis zu setzen, sodass Signale von ungebundener Sonde leichter ausgeschlossen werden konnten. Dies ermöglichte eine spezifische Markierung der T-Zellen. Es folgte die Synthese CB-markierter FIT-Sonden zur Aufklärung biologischer Fragestellungen, wie dem Verlauf einer Influenza A Infektion und die Synthese und Evaluation neuer Farbstoffe mit einem Absorptionsmaximum bei 590/596 nm. Zudem wurde der Einbau eines zyklischen PNA- Monomers bezüglich der Verbesserung von Responsivität und Helligkeit von PNA-FIT-Sonden analysiert. / In this work forced Intercalation (FIT) probes, developed by Seitz et al. were used for the mRNA characterization in living cells. The synthesis of improved FIT probes as well as the systematic study on the uptake of FIT probes by living Flp-In™ 293 T-REx™ cells was performed. Therefore FIT probe conjugates/aggregates as well as commercially available reagents, e.g. palmitic acid and the pore-forming enzyme Streptolysin-O were investigated under various conditions. Furthermore, the transfection was tested using an electroporator. The optimal transfection condition for the introduction of DNA and PNA FIT probes into Flp-In™ 293 T-REx™ cells was achieved using Streptolysin-O. Multicolor live cell imaging with the simultaneous use of three different FIT probes (BO, TO and QB) against three different target sequences was performed successfully. In addition, FIT probes were used for the differentiation between T cell lines. A competitive hybridization experiment with cells confirmed the specific fluorescence emission of the probes. Further studies with two cell lines and TO-FIT probes as well as terminal Cy7-labeled TO-FIT probes showed an increased TO emission in the presence of the complementary TCR mRNA target sequence in the cells. A second detection channel of the terminal Cy7 dye provided the advantage of comparing the Cy7- and TO-intensity ratio, thereby making it easier to exclude signals from unbound probe. This enabled the specific tagging of t cells. This was followed by the synthesis of QB-DNA-based FIT probes for the use in various biological applications e.g. as a pan selective marker for Influenza A infection. Moreover, the synthesis and evaluation of new dyes with an absorption maximum at 590/596 nm was performed. The incorporation of a cyclic PNA monomer next to the TO dye has also been realized to improve responsiveness and brightness in PNA-FIT probes.

No description available.