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Polymer flooding is one of the most used chemical methods for enhanced oil recovery(EOR). However, laboratory studies and field applications of polymer injections often encounter polymer-induced clogging due to polymer transport and entrapment, leading to permeability reduction and diminished recovery performance. In this work, we focus on understanding polymer flow behavior using microfluidics devices and fluorescence microscopy. Microfluidic devices were designed to mimic and replicate the pore-network structures of oil-bearing conventional reservoir rocks. We present various flow experiments to study polymer transport and the underlying mechanisms of polymer retention in porous media. We assess the polymer-induced clogging of partially hydrolyzed polyacrylamides - HPAMs, using tracers. Afterward, we use a commercially available fluorescent polymer with microfluidics and single-molecule microscopy to give insights into individual molecule dynamics. Furthermore, we perform numerical simulations to replicate and extend the experimental work. As these experiments were conducted using commercially fluorescent polymer of low molecular weight and due to limitations of tracers to track polymers, we extended this work to investigate the transport of HPAMs, which is the most used polymer for EOR, at molecule-scale. However, existent methods in the literature are not suitable for fluorescently labeling ultra-high molecule weight polymers. Therefore, we present a novel method for synthesis of dye-labeled polymers that successfully tagged the HPAMS. Finally, we assessed the conformation and flow dynamics of the fluorescently labeled HPAM molecules. The findings highlight a limitation in some polymer screening workflows in the industry that suggest selecting the candidate polymers based solely on their molecular size and the size distribution of the rock pore-throats. Moreover, we present, for the first time, direct visualization of the three main mechanisms underlying polymer retention in porous media. We bring the first molecular evidence of polymer pore-clogging and permeability reduction reversibility, which sheds light on the controversy in the literature. In addition, we propose a new method for fluorescent labeling water-soluble ultra-high molecular weight polyacrylamides-based polymers that preserves their viscosifying properties. The method can be extended to any polymers containing carboxyl groups or groups that can be functionalized into carboxyls, and therefore, the applicability covers any fields that employ polymers.

Bacteria typically adhere to various cell surfaces present in the human body to colonise or invade human tissues. Staphylococcus aureus (S. aureus) can express the fibronectin-binding proteins A and B (FnBP-A, FnBP-B) that can facilitate the binding of multiple copies of fibronectin (Fn). In addition, Fn bound to the bacterium trigger activation of α5β1 integrins found on the cells and facilitate invasion of human cells. Although the invasion mechanisms regarding signalling pathways and overall host cell interactions have been defined, the quantitative relationship between the mediators of invasion and the temporal kinetics has not yet been elucidated. In this thesis, newly developed microscopy-based methods have been used to quantify the interactions between H1299 cells and S. aureus at various Fn concentrations. After an approximate Fn concentration of 15 μg/ml, the S. aureus bacteria strains become saturated both for the wildtype and the negative control strains. Additionally, using the step-by-step protocol developed during this study, adhesion of the wildtype strain of S. aureus with 15 μg/ml Fn is occurring on the H1299 cells. Although adjustments to the protocol are needed, this adhesion mechanism will lead to an internalisation of the S. aureus strains to the H1299 cells.

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The development of new multiphoton absorbing materials has attracted the attention of researchers for the last two decades. The advantages that multiphoton absorbing materials offer, versus their one-photon absorbing counterparts, rely on the nature of the nonlinearity of the absorption process, where two photons are absorbed simultaneously offering increased 3D resolution, deeper penetration, and less photobleaching and photodamage as a result of a more confined excitation. The applications of efficient two-photon absorbing materials have been extensively expanding into the fields of photodynamic therapy, microscopy, and optical data storage. One of the fields where an increased interest in multiphoton absorbing materials has been most evident is in bioimaging, in particular, when different cellular processes and organelles need to be studied by fluorescence microscopy. The goal of this research was to develop efficient two-photon absorption (2PA) compounds to be used in fluorescence bioimaging, meaning that such compounds need to posses good optical properties, such as high fluorescence quantum yield, 2PA cross section, and photostability. In the first chapter of this dissertation, we describe the synthesis and structural characterization of a new series of fluorescent donor–acceptor and acceptor-acceptor molecules based on the fluorenyl ring system that incorporated functionalities such as alkynes and thiophene rings, through efficient Pd-catalyzed Sonogashira and Stille coupling reactions, in order to increase the length of the conjugation in our systems. These new molecules proved to have high two-photon absorption (2PA), and the effect of these functionalities on their 2PA cross section values was evaluated. Finally, their use in two-photon fluorescence microscopy (2PFM) imaging was demonstrated. iii One of the limitations of the compounds described in Chapter 1 was their poor water solubility; this issue was addressed in Chapter 2. The use of micelles in drug delivery has been shown to be an area of increasing interest over the last decade. In the bioimaging field, it is key to have dye molecules with a high degree of water solubility to enable cells to uptake the dye. By enclosing a hydrophobic dye in Pluronic® F-127 micelles, we developed a system that facilitates the use of 2PA molecules (typically hydrophobic) in biological systems for nonlinear biophotonic applications, specifically to image the lysosomes. Furthermore, we report in this chapter the efficient microwave-assisted synthesis of the dye used in this study. In addition, linear photophysical and photochemical parameters, two-photon absorption (2PA), and superfluorescence properties of the dye studied in Chapter 2, were investigated in Chapter 3. The steady-state absorption, fluorescence, and excitation anisotropy spectra of this dye were measured in several organic solvents and aqueous media. In Chapter 4, we describe the preparation and the use of an efficient and novel twophoton absorbing fluorescent probe conjugated to an antibody that confers selectivity towards the vascular endothelial growth factor receptor 2 (VEGFR-2) in porcine aortic endothelial cells that express this receptor (PAE-KDR). It is known that this receptor is overexpressed in certain cancer processes. Thus, targeting of this receptor will be useful to image the tumor vasculature. It was observed that when the dye was incubated with cells that do not express the receptor, no effective binding between the bioconjugate and the cells took place, resulting in very poor, nonspecific fluorescence images by both one and two-photon excitation. On the other hand, when the dye was incubated with cells that expressed VEGFR-2, efficient imaging of the cells was obtained, even at very low concentrations (0.4 μM). Moreover, incubation of the bioconjugate iv with tissue facilitated successful imaging of vasculature in mouse embryonic tissue

Im Laufe der Evolution haben Bakterienarten äußerst vielfältige und ausgeklügelte extrazelluläre Strukturen entwickelt, die es ihnen ermöglichen, Substrate in ihre Umgebung abzugeben oder Wirtszellen während einer Invasion anzugreifen. Diese Sekretionssysteme sind an vielen bakteriellen Mechanismen wie Biofilmbildung, Zellmotilität, Virulenz oder Gentransfer und Verbreitung von Antibiotikaresistenzen beteiligt. Das Verständnis des Aufbaus und der Regulierung dieser Strukturen ist angesichts der zunehmenden Entwicklung multiresistenter Bakterien von entscheidender Bedeutung. Darüber hinaus geht der Aufbau solcher Strukturen unweigerlich auf Kosten wertvoller zellulärer Energieressourcen, was einen spannenden Parameter darstellt, um zu untersuchen, wie Bakterien den optimalen Mechanismus zum Ausgleich zwischen zellulären Mechanismen und Energieverbrauch wählen. Diese Arbeit konzentriert sich auf den Aufbau und die Regulierung bakterieller extrazellulärer Filamente, insbesondere des flagellaren Typ-III-Sekretionssystems (T3SS). Im ersten Kapitel werden Defekte in der Zellmorphologie aufgezeigt, die durch die Deletion des FlhE-Proteins während des Zusammenbaus der Flagellen verursacht werden, was die Bedeutung der Regulierung des Membranpotentials verdeutlicht. Das zweite Kapitel zeigt, dass der Assemblierungsmechanismus der Flagellen-Filamente, welcher dem Injektions-Diffusions-Modell entspricht, im Vergleich zu anderen Sekretionssystemen schneller ist und für die Energieerhaltung optimiert ist. Das dritte Kapitel untersucht die Rolle des Pilus beim Plasmid-Transfer, der mit einem Typ-IV-Sekretionssystem (T4SS) assoziiert ist, und liefert zusätzliche Hinweise darauf, dass der Pilus als Kanal für den Plasmid-DNA-Transfer dienen kann. Im letzten Kapitel wird ein neuer Biosensor zur Messung des Gehalts an bis-(3'-5')-zyklischem Diguanosinmonophosphat (c-di-GMP) entwickelt, einem entscheidenden Molekül in bakteriellen Mechanismen, die Zellmotilität und Biofilmbildung miteinander verbinden. Insgesamt bietet diese Arbeit Einblicke in die Regulation des flagellaren T3SS und des T4SS-Pilus, ein neues Werkzeug zur Untersuchung von c-di-GMP und Einblicke, wie Bakterien entscheidende Überlebensparameter und die Optimierung eines energiesparenden Aufbaus abwägen. / Through evolution, bacterial species have developed highly diverse and sophisticated extracellular structures enabling them to secrete substrates in their environment or to target host cells during invasion. Those secretion systems are involved in many bacterial mechanisms such as biofilm formation, cell motility, virulence or gene transfer and antibiotic resistance dissemination. Understanding the assembly and regulation of these structures is crucial due to the increasing development of multi-drug resistant bacteria. Moreover, the assembly of such structures inevitably comes at the cost of valuable cellular energy resources, representing an exciting parameter to study how bacteria selected the optimal mechanism to balance cellular mechanisms and energy consumption. This thesis focuses on the assembly and regulation of bacterial extracellular filaments, notably the flagellar type III secretion system (T3SS) flagellum . The first chapter reveals cell morphology defects caused by the deletion of the FlhE protein during flagellum assembly, highlighting the importance of membrane potential regulation . Chapter two illustrates that the flagellar filament assembly mechanism, following the injection-diffusion model, is faster compared to other secretion systems and optimized for energy conservation. The third chapter investigates the role of the pilus in plasmid transfer, associated with a type IV secretion system (T4SS), and gives additional evidence that the pilus may also act as a channel for plasmid DNA transfer. The final chapter develops a new biosensor for measuring bis-(3’-5’)-cyclic diguanosine monophosphate (c-di-GMP) levels, a crucial molecule in bacterial mechanisms linking cell motility and biofilm formation. Overall, this thesis provides insights into the regulation of the flagellar T3SS and the T4SS pilus, a new tool to study c-di-GMP, and how bacteria balance crucial survival parameters and energy-conserving assembly optimization .

Biomolecular condensates are dynamic intracellular structural units or distinct reaction spaces that can form by condensation of their constituents from the cytoplasm or the nucleoplasm. It is generally not clear yet, how dynamic, continuum-like condensate properties relevant for large-scale intracellular organisation emerge from the interplay of proteins and nucleic acids on the level of few individual molecules. With this work, we expand the portfolio of methods to investigate the role of protein-nucleic acid interactions in biomolecular condensates by introducing optical tweezers-based mechanical micromanipulation of single DNA molecules combined with confocal fluorescence microscopy to the field. We used this approach to characterise how the two landmark proteins1 Fused in Sarcoma and Heterochromatin Protein 1 form condensates with single DNA molecules. Fused in Sarcoma (FUS) is a key protein for various aspects of the nucleic acid metabolism and evidence is accumulating that biomolecular condensation is crucial for both, its physiological functions and its role in pathological aggregate formation. In this thesis, we directly visualised the formation of FUS condensates with single molecules of ssDNA and dsDNA. We showed that the formation of these microcondensates is based on nucleic acid scaffolding. We explored their mechanical properties and found that the mechanical tension that (FUS dsDNA) condensates can withstand or exert is in the range below 2 pN. We further demonstrated that already on this fundamental scale and with limited amounts of constituent molecules, dynamic properties like shape relaxations, reminiscent of viscoelastic materials, can emerge. Heterochromatin Protein 1 (HP1) is a prototypic chromatin organising factor that is in particular involved in the formation of dynamically compacted heterochromatin domains. HP1 forms biomolecular condensates and compacts DNA strands in vitro. In this work, we measured the influence of HP1 on the mechanical properties of individual DNA molecules and demonstrated the response of HP1-DNA condensates to different environmental conditions. We contributed a methodological framework to characterise viscoelastic-like systems on the single molecule level. Taken together, our optical tweezers-based approach revealed structural and mechanical properties of prototypic protein-DNA condensates and hence helped to elucidate mechanisms underlying their formation in unprecedented spatiotemporal and mechanical detail. We anticipate that this method can become a valuable tool to investigate how large-scale intracellular organisation based on protein-nucleic acid condensation emerges from interactions between individual building blocks.

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