FtsH is a universally conserved, membrane-bound AAA+ protease critical for the quality control of membrane proteins. While the nanomechanics of homologous proteases have been widely studied, the nanomechanical profile of FtsH, remains largely unexplored. This thesis investigates the hyperthermophilic FtsH variant from Aquifex aeolicus using single trap optical tweezers.
First, we validated the functional integrity of detergent-solubilized AaFtsH through bulk biochemical assays. Wedemonstrated robust proteolytic activity against both disordered (β-casein) and structured (titin-I27 variants) at the experimental optical trapping temperature of 50°C.
To distinguish sub-nanometer motor steps from the high thermal fluctuations and instrumental drift inherent to high-temperature experiments, we conducted a rigorous system noise analysis and validation. We demonstrated that standard step-finding algorithms can be optimized through a novel Allan Variance-based Kalafut-Visscher step-fitting (AVKV) method and released it as an open-source Python toolkit. This workflow objectively determines optimal resampling bandwidth, ensuring that step detection is driven by statistical rigor.
Using this validated AVKV framework, we resolved distinct translocation events by FtsH character ized by a mean step size of 1.15±0.64 nm and a step dwell time of 0.29±0.28 s. Notably, repeated unfolding patterns were not observed for the titin-V13P substrate, likely due to a combination of mutation and elevated temperature rendering the substrate unstable.
Collectively, this work provides the first single-molecule observation of processive stepping by AaFtsH and establishes a standardized, reproducible computational framework for analyzing optical tweezers data in high-noise environments.

Lipid bilayers are fundamental to cell membranes. This thesis develops novel opto-microfluidic platforms for studying artificial cell membranes, addressing long-standing experimental challenges. Utilizing the developed tools, the work investigates thermomechanical properties of phase-separated membranes, the impact of lipid tail-asymmetry and cholesterol on mechanical properties, electrophysiological measurements on freestanding bilayers, and the effect of azithromycin on bilayers. This research provides new insights into membrane biophysics and advances drug-screening capabilities.

The role of ribosome-bound chaperones and nascent chain interactions in protein biogenesis holds many open questions. This thesis provides new insights into the intricate mechanisms governing co-translational folding and complex assembly. By combining in vivo genome-wide screening results from selective ribosome and disome selective profiling, conducted by our collaborators B. Bukau and G. Kramer at Heidelberg University, with our in vitro single-molecule force spectroscopy and correlated confocal imaging results, we not only gained a better understanding of the general prevalence, but also of the mechanistic details of these cellular processes.

We investigated how Trigger Factor (TF), the only ribosome-associated chaperone in bacteria, influences nascent chain folding of a single-domain protein. We show for the first time that TF accelerates folding on the ribosome. This process is regulated by translation, with the emergence of key peptide segments dictating TF’s ability to compact nascent chains and stabilize partial folds. Beyond chaperone-modulated folding, we also explored how ribosome cooperation drives protein complex formation. Using the intermediate filament lamin as a model, we demonstrate that ribosome proximity enables nascent chains to ‘chaperone each other,’ facilitating coiled-coil formation while preventing misfolding. Notably, when early interactions between nascent chains are inhibited or delayed, they become trapped in misfolded states and are no longer assembly-competent. We further examined the role of timing in nascent chain interactions, using the BTB domain as a model to study the challenging formation of intertwined dimers during translation. We identify a translation-driven temporal control mechanism that ensures proper dimerization. This process opens otherwise inaccessible folding-assembly pathways, bypassing unproductive monomeric states.

Taken together, we have managed, through the powerful combination of in vivo and in vitro approaches, to gain new perspectives on how cells ensure faithful protein biogenesis.

Incorporating living cells into a non-living matrix is one of the many possible steps that can be undertaken to stop climate change. Especially, photosynthetic organisms have a promising future in material design as they can capture atmospheric carbon dioxide and ’ breathe’ oxygen. This research dives into unravelling the properties of a hydrogel-based living material containing C. reinhardtii.
To facilitate a systematic exploration, this goal was divided into three smaller pieces. Firstly, the study delves into the mechanical characteristics, aiming to identify the most suitable bio-ink crosslink technique and composition. To validate the mechanical properties the living material was subjected to rheology and a bridging test. Concluded can be that crosslinking and algae growth improve the mechanical stability of the material, whereas, gelatin did not. The sagging behaviour of the material looked promising.
Secondly, the photosynthetic activity of this living material was researched. It was found that the rise of O2 levels can not be measured accurately, the non-living matrix can release a high amount of CO2 of over 20.000 ppm and airtightness poses a complex challenge in this field of research.
Lastly, the project incorporates attempts to find effective techniques for studying the livingness of this unique material. In this part of the research, inverted optical microscopy, 3D laser scanning microscopy and chlorophyll extraction were discarded as suitable methods to study the livingness of the algae material. It was proven that leveraging the autofluorescence of the algal chlorophyll confocal laser scanning microscopy gives high-resolution images and the livingness of this living material could be studied with this technique in the near future.
This research significantly contributes to our understanding of this hydrogel-based living material and its many challenging properties. It underscores the importance of innovative materials like these in addressing contemporary environmental challenges, particularly in carbon capture. Moreover, it highlights the complexity of characterizing such materials, paving the way for further exploration and development in this relatively new field.

Everything that we consider alive, be it plants, dogs, bacteria or humans, is composed of the same microscopic building blocks: cells. While cells between and even within these organisms can look and behave very differently, they all share the same key functionalities: they can grow, they can divide to proliferate, they can eat and metabolise to fuel internal processes, and they carry a genetic blueprint which they can process to knowwhat to do and how to do it. Since the first notion that cells formthe universal building blocks of life [1], biologists have been intrigued by the fact that across the wide diversity of living organisms, all these constituent cells share the same fundamental characteristics. In the process of understanding how cells are capable of executing the key functionalities of growth, division, metabolism and information processing, biologists have identified the set of molecular components that constitute cellular life, and have broadly related components to specific cell functions. Despite the growing knowledge on what there is inside the cell, a crucial question remains largely unanswered: how do the molecular components of a cell give rise to these life-giving processes? Answering that question that is easier said than done. Cells consist of thousands of different components that are in continuous interaction, can take over each other’s function, or have multiple functions. Extracting a mechanistic understanding of how these components actually work together to make the cell alive, is almost impossible in such a complex soup. Even if we can list the minimal set of components that are vital to cellular life, we gain little understanding in how these ingredients function to make non-living matter alive. Instead, for this purpose it might be more insightful to try to rebuild the cell with a minimal set of components: to build a synthetic cell. In such a bottom-up reconstitution approach, cell components and their interactions can be studied in a well-controlled chemical environment. Starting with a small number of components, complexity can step-by-step be increased while maintaining a fundamental understanding throughout the construction process. To achieve this goal of building a minimal cell, multiple research initiatives have been founded worldwide. As a unique interdisciplinary effort, synthetic cell research combines the expertise required to understand, rebuild and integrate all vital cellular functions in vitro...

This project started with analyzing the different roles and potentials that bacteria have for the growth of new materials with ecological benefits. Here questions arise about the role that a designer can fulfill in the development of such novel materials. What new skillset does he/she need to obtain? How will a collaboration with biologists take place? In order to get a better understanding of the potentials of materials grown by bacteria and the subsequent role of a designer, a Material Driven Design (Karana et al., 2015) project was performed in collaboration with scientists from the Aubin-Tam research group, part of the Bionanoscience department of the Delft University of Technology. The starting point of the design project was a composite material consisting of three ingredients that are grown by three separate species of bacteria. In order to gain an understanding of how this material was grown and produced, the designer performed a plethora of experiments investigating; the growth of the organisms and the amount of material they produced; how the ratio between the three ingredients influenced the resulting material; how the way in which the material was processed resulted in its final form and properties. This led to the understanding that this material is highly programmable in its form and properties such as its flexibility, strength and surface roughness. With this in mind, user studies were performed in which it was found that the versatility of the material was considered interesting and intriguing by participants. They wonder what it is and how it is made, finding it hard to believe that bacteria grew such a material. This led to a material concept in which the designer proposes to play with these varying properties of the material, resulting in contrasting material experiences and highlighting the material its ability to appear as something that is both natural and man-made at the same time. This was done by exploring various processesing potentials of the material and analysing how different parameters of these processes influence the resulting material its properties. In doing so, the designer provided a framework by which future designers can program and explore this bio-based material that shows a lot of different potentials.

The microscopicworld is surprisingly busywith swimming micro-organisms. In a droplet of pondwater, there can be tens of thousands of microbes. They are predators and preys, producers and consumers, and they formthe bottom levels of the ecology of our world. The swimming micro-organisms can be in general classified into prokaryotes and eukaryotes. Eukaryotes come later in evolution. They are higher organisms than the prokaryotes as they possess more complex cellular structures such as the nucleus and mitochondria. Motile eukaryotic micro-organisms all use an active hair-like structure for swimming, known as flagella or cilia. Although nuanced difference exists between flagella (cilia) of different species, their general internal structure and driving mechanisms are mostly the same. In some sense, they are one of the bestselling machines for locomotion on themicron scale. Although flagella are the first-ever documented organelles in cell biology, our understanding of them is still limited. For example, we have only begun to appreciate how the conformational change of single protein motors results in the waveformon the scale of a flagellum. Our understanding of the flow generated by even a single flagellum is rudimentary: resolving the temporal features of such flow field remains experimentally challenging. On a larger scale, how thousands of cilia interact with each other to facilitate fluid transport is still elusive: theoretical models and simulations are waiting for experimental verification. In this thesis, I explore different topics centering around flagellar/ciliary motility by employing novel experimental and numerical techniques, and hence advance our understanding. My experimental investigation starts by resolving the flow generated by the beating cilia of single cells. Due to the high beating frequency, high temporal resolution is required to map the time-varying flow field, which conventional tracer particle-based flow velocimetry techniques cannot provide. To tackle this challenge, I implemented an optical tweezers-based flow velocimetry (OTV) technique. In this technique, a bead is trapped and placed at a particular location by a focused laser beam. The local flow displaces the bead from the trapping center. This displacement, although small, can be accurately monitored by laser interferometry and converted into an electrical signal by photoelectric detectors. Essentially,we gain the desired accuracy and temporal resolution by exploiting the high resolution and large bandwidth of interferometric and electrical measurements. With this technique, I revealed that the ciliary flow deviates fundamentally fromhow it is often modeled by Stokes equations. More specifically, the flow’s amplitude decays faster spatially, and its phase shifts over distance. These discrepancies are resolved by adding a linear unsteady term to Stokes equations. Furthermore, I systematically characterized the ciliary flow field created by captured C. reinhardtii cells. The flow field in different directions and over the ciliary beating plane are measured experimentally, modelled numerically, and analyzed theoretically. Results displayed excellent agreement with each other, and altogether increased our knowledge in the ciliary flow. With the OTV measurements, I not only studied the basic hydrodynamics of ciliary flowbut also addressed a long-standing hypothesis regarding the function of a ciliary appendage. Many cilia have fibrous ultrastructures called mastigoneme. These fibrous appendages are believed to help cells swim faster by increasing the ciliary surface area. Our experiments, together with numerical studies, completely refute this hypothesis: such fibrous hairs do not show any hydrodynamic significance in C. reinhardtii. Instead, its absence in genetically modifiedmutants appeared to result in some behavioral changes, causing the cells to turn abruptly more often than usual. Therefore, I have re-opened the question about the function of the fibrous mastigonemes. Future investigation towards this direction is needed and is likely to lead to more exciting findings. Lastly, I attempted to bridge the physics of ciliary flowwith the biology of ciliary beating. I focused on the ciliary difference and investigated it by selectively loading each cilium of C. reinhardtii with external flows. The ciliary difference is critical for the steering of biflagellates (micro-organisms swimming with two flagella/cilia). I observed an unreported functional difference between the two cilia, as I found that the coupling between the two cilia is unilateral. One cilium serves as the coordinator of beating, and a cell is coupled to external hydrodynamic forces mostly through this coordinating cilium. Altogether, by introducing the OTV technique and incorporating different numerical methods, I was able to elucidate the ciliary flowin a time-resolvedway, updating the current understanding in these unsteady flows. The effectiveness of this methodology was demonstrated again by its application in studying the function of the fibrous ultrastructures. By further moving on to the biological aspect of ciliary beating, we found a new type of difference between the two cilia, which enriches our knowledge in inter-ciliary coupling.

In this study, a method for the bacterial production of a nacre-mimicking composite material was developed. Nacre (mother-of-pearl) is an organic-inorganic composite found in the inner lining of many mollusk shells and in pearls. It has a brick-and-mortar structure consisting of 95% aragonite (calcium carbonate) platelets and 5% organic matrix. Serving as a protective structure against e.g. predators, nacre has developed into an extremely strong and tough material, despite largely consisting of ceramic calciumcarbonate. Numerous mechanisms have been proposed to explain the outstanding mechanical properties of nacre, such as crack deflection and local strain hardening. Many groups are pursuing the aim of developing new materials which mimic nacre’s structure and mechanical properties. Nacre is produced by mollusks at ambient temperatures with easily obtainable materials and with low expenditure of energy. In contrast, human methods usually require extensive energy input, high temperatures and/or pressures, and environmentally damaging chemicals.

Proteins are one of the most important constituents of cells, since they are responsible for various processes such as replication, translation, etc. Interestingly, proteins are also responsible for the degradation of proteins that do not assemble well, and/or which are not necessary anymore inside the cell. This process of proteins destruction and recycling of amino acids is called proteolysis. Without this process, the cell would accumulate toxic waste and would not survive for long. This highlights the importance of this process. This process occurs not only in the cell cytoplasm but in the cell lipid bilayer leading to the degradation of soluble and membrane integral proteins. In bacteria, the protein responsible for this mechanism is vital and it is called FtsH. FtsH is part of the AAA+ proteases family, which are deeply investigated since they are responsible for many important biological mechanisms inside the cell.
In this thesis, we aimed to answer the question of how can FtsH not only degrade soluble proteins, but also degrade insoluble proteins. In a complex mechanism in which soluble/insoluble proteins are unfolded passing through an ATPase domain, and into a protease domain for degradation. The curiosity about this mechanism is also high, regarding how can this protein coordinate this ATP hydrolysation and coordinate it with the proteolytical process.
To answer these questions, this thesis presents a series of purification protocols for E. coli FtsH (Chapter 2) and for an orthologous of FtsH, a thermophile called Aquifex aeolicus (presented in Chapter 3). During this thesis, we show that the movements that the ATPase domain can undergo in relation to the membrane are larger than what was previously described in literature (Chapter 4). The assembling of this protein into dodecamers, in the solubilized form, showed that the intermembrane loops are more flexible than what was thought before. A kinetic characterization of the ATPase and protease activity is also assessed showing that both forms are equally functional. Finally, in Chapter 5, we explore the use of cryo-electron microscopy and tomography to perform an exhaustive single particle study. Although the cryo-TEM results showed in this chapter are preliminary 2D class averages, it is possible to observe the six-fold symmetry structure of this protein, which is an incentive to pursue the studies with this technique. The same is true for the cryo-tomography performed on FtsH in the proteoliposomes which will provide further insights about the protein insertion into the membrane and allow to the study how substrates can access the ATPase domain loops.
This thesis describes the efforts made in the FtsH purification protocol optimization to get a sample as pure and stable as possible. This thesis showed that FtsH undergoes much larger conformational changes than previously thought and challenges the currently accepted model for the substrate to access FtsH active site.
In the future cryo-electron microscopy of single particles and cryo-tomography of proteoliposomes must be explored too deepen our knowledge of the full-length FtsH structure, and more generally our knowledge about proteolytical mechanisms in cells.