Analysis of sub-10 nm precipitates extracted from microalloyed steels

Microalloyed steels contain small quantities (≤ 0.5 wt%) of the microalloying elements Ti, Nb, V. Judicious combination of TMCP parameters and microalloyed steel composition leads to formation of desirable nm-sized carbide, nitride, carbonitride inclusions which improve steel mechanical properties. TMCP optimisation relies on understanding the interrelation between TMCP parameters and precipitate properties. A characterisation routine was developed in the group to provide statistically meaningful data on precipitates size distribution and chemical composition.[1] Precipitates with diameters below 10nm could not be investigated with the existing routine. Such precipitates are of interest because they play a key role in precipitation hardening. This thesis extends the existing characterisation routine to sub-10nm precipitates extracted from microalloyed steels. Electrolytic extraction was investigated as alternative extraction process to reduce undesired particle loss during chemical extraction. The suitability of various electrolytes to provide a stable colloidal suspension for colloidal analysis was assessed. Chemically extracted precipitates underwent differential centrifugation to isolate sub-10nm precipitates and enable their size and chemical composition characterisation. Improvements in precipitate analysis were achieved by implementation of speed-ramp analytical ultracentrifugation and precipitate number density determination.Mikrolegierte Stähle enthalten geringe Mengen (≤ 0.5 wt%) der Mikrolegierungselemente Ti, Nb, V. Eine geschickte Kombination aus TMCP-Parametern und Stahlzusammensetzung führt zur Bildung nm-großer Karbid-, Nitrid-, Karbonitrideinschlüsse, die mechanische Eigenschaften verbessern. TMCP-Optimierung beruht auf dem Verständnis des Zusammenhangs zwischen TMCP-Parametern und Ausscheidungseigenschaften. Eine vorhandene Charakterisierungsroutine liefert statistisch aussagekräftige Daten über die Größenverteilung und die chemische Zusammensetzung der Ausscheidungen.[ 1] Ausscheidungen mit Durchmessern unter 10nm konnten mit der bestehenden Methode nicht untersucht werden. Solche Ausscheidungen sind von Interesse, da sie eine Schlüsselrolle bei der Ausscheidungshärtung spielen. Diese Arbeit erweitert die bestehende Charakterisierungsroutine auf extrahierte Ausscheidungen kleiner 10 nm. Elektrolytische Extraktion wurde als alternatives Extraktionsverfahren erprobt, um unerwünschten Partikelverlust zu reduzieren. Die Eignung von Elektrolyten zur Bereitstellung einer stabilen kolloidalen Suspension zur Analyse mit kolloidalen Messmethoden wurde bewertet. Fraktionierung chemisch extrahierter Ausscheidungen mittels Differentialzentrifugation ermöglichte die Charakterisierung von Ausscheidungen kleiner 10 nm. Ausscheidungscharakterisierungsverbesserungen wurden durch Einführung der analytischen Ultrazentrifugation mit Geschwindigkeitsrampe und der Bestimmung der Ausscheidungsdichte erzielt

Characterization, Manipulation, and Prediction of Protein Aggregation in Model Systems

Main influencing factors favoring protein aggregation are to be identified regarding solution conditions of the surrounding aqueous solution and regarding physicochemical properties of the model proteins themselves. Based on these experimental results, concepts are developed for manipulation of protein aggregation behavior. Finally, based on selected parameters that significantly impact protein aggregation behavior, approaches are developed to predict protein aggregation propensity

Development of High Throughput Screening Methods for the Automated Optimization of Inclusion Body Protein Refolding Processes

In this study robotic HTS methods for protein refolding were developed with a focus on HTS compatible analytics. Solubility was identified as a meaningful objective function during refolding processes. Measurement of protein solubility in a highly complex buffer matrix with varying protein conformations was established. A genetic algorithm in combination with DoE was used for an effective and fully automated optimization of 5 process parameters in parallel in a wide parameters space

Bacteria Hunt: Evaluating multi-paradigm BCI interaction

The multimodal, multi-paradigm brain-computer interfacing (BCI) game Bacteria Hunt was used to evaluate two aspects of BCI interaction in a gaming context. One goal was to examine the effect of feedback on the ability of the user to manipulate his mental state of relaxation. This was done by having one condition in which the subject played the game with real feedback, and another with sham feedback. The feedback did not seem to affect the game experience (such as sense of control and tension) or the objective indicators of relaxation, alpha activity and heart rate. The results are discussed with regard to clinical neurofeedback studies. The second goal was to look into possible interactions between the two BCI paradigms used in the game: steady-state visually-evoked potentials (SSVEP) as an indicator of concentration, and alpha activity as a measure of relaxation. SSVEP stimulation activates the cortex and can thus block the alpha rhythm. Despite this effect, subjects were able to keep their alpha power up, in compliance with the instructed relaxation task. In addition to the main goals, a new SSVEP detection algorithm was developed and evaluated

Controllable Acoustic Mixing of Fluids in Microchannels for the Fabrication of Therapeutic Nanoparticles

Fifteen years ago, surface acoustic waves (SAW) were found to be able to drive fluids and numerous applications in microfluidics have been developed since. Here, we review the progress made and report on new approaches in setting-up microfluidic, continuous flow acoustic mixing. In a microchannel, chaotic advection is achieved by generation of a SAW driven fluid jet perpendicular to the mean flow direction. Using a high speed video camera and particle image velocimetry, we measure the flow velocities and show that mixing is achieved in a particularly controllable and fast way. The mixing quality is determined as a function of system parameters: SAW power, volume flux and fluid viscosity. Exploring the parameter space of mixing provides a practical guide for acoustic mixing in microchannels and allows for adopting conditions to different solvents, as e.g., required for the generation of nanoscale particles from alcoholic phases. We exemplarily demonstrate the potential of SAW based continuous flow mixing for the production of therapeutic nucleic acid nanoparticles assembled from polymer and lipid solutions

Hemithioindigos for cellular tubulin photopharmacology: From proof of concept to near-quantitatively photoswitchable tools with single-cell precision

Microtubules (MTs) are hollow polymeric tubes formed of α/β-tubulin heterodimers that are in a constant dynamic equilibrium between growth and shrinkage. MTs are an essential constituent of the cytoskeleton and so are critical in many cellular processes, such as mitosis, cargo trafficking and cell migration. Consequentially, modulation of MT dynamics is a promising objective in the development of biological research tools, as well as therapeutic drugs. However, since MTs fulfil many different physiological roles at the same time, global manipulation of the MT network often causes serious off-site effects. This poses a major challenge in MT biology research. A major goal is thus to develop MT-targeting research tools whose activity can be precisely controlled: in terms of location (spatial) to selectively address the area under study, and at a given time point (temporal). Owing to its unrivalled precision, non-invasiveness, and reversibility, light is an almost ideal stimulus for such spatiotemporally precise modulation of bioactivity. Photopharmacology harnesses these unique features of light to create high-precision photopharmaceuticals. Reversible photopharmaceuticals are bioactive small molecules derivatized with a molecular photoswitch that has two photoisomers that differ in binding affinity towards the biological target. Driven by light, these two isomers are interconverted between each other, thus enabling optical control over protein activity. Previous approaches towards optical control over MT dynamics have mainly been based on azobenzene-derived photopharmaceuticals. However, azobenzenes are by no means ideal photoswitches, in that they suffer from the need of potentially phototoxic UV light, incomplete photoisomerization, incompatibility with certain functional groups and metabolic lability, among others. Therefore, this thesis aims at the development of MT photopharmaceuticals that overcome some of the limitations and weaknesses of azobenzene-based approaches. These photopharmaceuticals are based on hemithioindigo (HTI), an emerging photoswitch with various beneficial properties, whose use in photopharmacology has however been scarce and far from systematical. In the following, I describe the development of hemithioindigo-based photopharmaceuticals for MT biology. Paper One demonstrates the first use of HTIs in cellular photopharmacology (HOTubs). We showcase their potential for long-term photocontrol over the MT network, and their suitability for photopharmacology in general. We show in live cells that these HTIs light-dependently induce cytotoxicity, disruption of the MT network and mitotic cell cycle arrest. They also bring unique possibilities: for most photoswitches including azobenzenes, isomerization is accompanied by large changes in sterics and consequentially it is often the sterics of the protein binding site that decides which photoisomer is the stronger binding one; but since both HTI isomers are nearly planar, we could use rational design rather than protein sterics to control which of the two HOTub isomers was the more bioactive. Taken together with the absence of unspecific toxicity and the avoidance of potentially phototoxic UV light, our findings strongly recommend HTIs as general and cell-compatible scaffolds for photopharmacology. Paper Two aims at increasing the cellular potency of our HTI-based tubulin photopharmaceuticals. I design a SAR study and outline how the structural similarity of a highly potent indanone and hemithioindigo can be harnessed to create research tools (HITubs) exceeding the potency of all MT photopharmaceuticals known at the time. Another key aspect of this paper is to show that, unlike azobenzenes, the metastable isomer of HTI photopharmaceuticals has slow enough relaxation even with a strongly electron-donating, tautomerizable -OH group in the para-position, to allow its use as a bistable photoswitch. This substantially widens the chemical scope of photopharmaceuticals and thus expands the range of biological targets addressable by photopharmacology. Paper Three describes how structural finetuning of the photoswitch scaffold yields MT photopharmaceuticals with near-quantitative photoisomerization (PHTubs). This enables studies void of undesired background of bioactivity which would stem from incomplete photoisomerization. For this purpose, I design a SAR study and synthesize tools based on pyrrole hemithioindigo (PHT), a photoswitch closely related to HTI. We further show that our PHTubs are sturdy photopharmaceuticals, that unlike azobenzenes, resist glutathione-mediated degradation in both isomeric states. This is an important prerequisite for more advanced studies, and we illustrate how PHTs can be implemented to light-dependently modulate the MT network in living cells and with single-cell precision. Beside these topics, I briefly present my unpublished work on two other projects. The first aims for optical control over γ-tubulin, an isoform involved in cellular MT nucleation. Taking advantage of the similarity between an isoflavone-based γ-tubulin specific inhibitor and HTI, I design an HTI-based photopharmaceutical thought to target MT nucleation (phatastatin). We are currently optimizing a key step en route to phatastatin. The second project aims towards optical control over proteasomal degradation of tubulin. In the context of a research stay in the Trauner lab at New York University (NYU), I designed and synthesized a series of photoswitchable tubulin degraders (TubPHOTACs), and we are currently evaluating two promising compounds for their cellular mechanism. In summary, this work contributes to light-dependent modulation of MT dynamics and introduces indigoid photoswitches (HTI and PHT) as sturdy photoswitches allowing for visible-light powered, reversible and highly precise studies in photopharmacology