Artificial Metalloenzymes

In de natuur wordt een groot deel van de chemische transformaties uitgevoerd door complexe moleculen, die bekend staan als enzymen. Deze enzymen zijn katalysatoren die vaak bouwstenen produceren van één spiegelbeeld. Het produceren van bouwstenen van slechts een spiegelbeeld is belangrijk voor het maken van bijvoorbeeld medicijnen. Echter natuurlijke enzymen kunnen slechts een gelimiteerde set van deze transformaties uitvoeren. Homogene katalyse, vaak gebruikt in organische chemie, kan een veel bredere set aan transformatie uitvoeren, maar niet altijd van één spiegelbeeld. Artificiële metaalenzymen maken gebruik van de voordelen van de twee eerder genoemde katalysatoren en combineert deze tot een. Zo’n katalysator bestaat uit een biomolecuul en een metaalcomplex. Toepassing van deze katalysatoren is al zeer succesvol gebleken, echter het aantal biomoleculen dat beschikbaar is voor de constructie van artificiële metaalenzymen is beperkt.Dit proefschrift beschrijft een nieuwe benadering voor ontwikkeling en constructie van artificiële metaalenzymen.Een actieve centrum, waar de chemische transformatie plaats vindt, werd gecreëerd op een interface van een eiwit dat bestaat uit twee gelijk delen. Hiervoor werd het eiwit LmrR gebruikt. Twee benaderingen om het metaal complex te verankeren aan LmrR werden gedemonstreerd, namelijk op een covalente en niet covalente manier. De artificiële metaalenzymen die hieruit ontstonden, konden verschillende katalytische transformaties uitvoeren waarbij zeer goede resultaten werden behaald. Dat wil zeggen dat een hoog percentage van een spiegelbeeld werden gevormd. Een verdere studie van deze katalysatoren gaf een beter inzicht hoe deze transformaties uitgevoerd werden.In nature, a large scope of chemical transformations are performed by complex molecules, known as enzymes. Enzymes are catalysts that are able to produce building blocks of just one mirror image. The production of these building blocks possessing just one mirror image is important for the construction of medicine, for example. However, enzymes have a very limited scope of transformation they can achieve. On the other hand, homogenous catalysis, can perform a much wider scope of transformations. However, it does not always produce one mirror image of the building block. Artificial metalloenzymes utilizes the benefits of the catalysts mentioned previously. An artificial metalloenzymes consists of a metal complex and a biomolecule. Artificial metalloenzymes proved to be very successful, however the number of available biomolecules for the construction of artificial metalloenzymes is limited.This thesis describes a new approach for the development and construction of artificial metalloenzymes.An active site, in which the chemical transformation takes place, was constructed on the interface of a protein that consisted of two equal parts. The protein that was used was LmrR. Different approaches to anchor the metal complex covalent or non-covalent are described in this thesis. The resulting artificial metalloenzymes were able to catalyze a variety of chemical transformations with very good results, i.e. a high percentage of one of the two mirror images of the products were formed. A study of these artificial metalloenzyme gave more insight in how these transformations were performed

Artificial Metalloenzymes

In de natuur wordt een groot deel van de chemische transformaties uitgevoerd door complexe moleculen, die bekend staan als enzymen. Deze enzymen zijn katalysatoren die vaak bouwstenen produceren van één spiegelbeeld. Het produceren van bouwstenen van slechts een spiegelbeeld is belangrijk voor het maken van bijvoorbeeld medicijnen. Echter natuurlijke enzymen kunnen slechts een gelimiteerde set van deze transformaties uitvoeren. Homogene katalyse, vaak gebruikt in organische chemie, kan een veel bredere set aan transformatie uitvoeren, maar niet altijd van één spiegelbeeld. Artificiële metaalenzymen maken gebruik van de voordelen van de twee eerder genoemde katalysatoren en combineert deze tot een. Zo’n katalysator bestaat uit een biomolecuul en een metaalcomplex. Toepassing van deze katalysatoren is al zeer succesvol gebleken, echter het aantal biomoleculen dat beschikbaar is voor de constructie van artificiële metaalenzymen is beperkt. Dit proefschrift beschrijft een nieuwe benadering voor ontwikkeling en constructie van artificiële metaalenzymen. Een actieve centrum, waar de chemische transformatie plaats vindt, werd gecreëerd op een interface van een eiwit dat bestaat uit twee gelijk delen. Hiervoor werd het eiwit LmrR gebruikt. Twee benaderingen om het metaal complex te verankeren aan LmrR werden gedemonstreerd, namelijk op een covalente en niet covalente manier. De artificiële metaalenzymen die hieruit ontstonden, konden verschillende katalytische transformaties uitvoeren waarbij zeer goede resultaten werden behaald. Dat wil zeggen dat een hoog percentage van een spiegelbeeld werden gevormd. Een verdere studie van deze katalysatoren gaf een beter inzicht hoe deze transformaties uitgevoerd werden

Diagnostic, Prognostic, and Therapeutic Implications of Genetic Testing for Hypertrophic Cardiomyopathy

Over the last 2 decades, the pathogenic basis for the most common heritable cardiovascular disease, hypertrophic cardiomyopathy (HCM), has been investigated extensively. Affecting approximately 1 in 500 individuals, HCM is the most common cause of sudden death in young athletes. In recent years, genomic medicine has been moving from the bench to the bedside throughout all medical disciplines including cardiology. Now, genomic medicine has entered clinical practice as it pertains to the evaluation and management of patients with HCM. The continuous research and discoveries of new HCM susceptibility genes, the growing amount of data from genotype-phenotype correlation studies, and the introduction of commercially available genetic tests for HCM make it essential that the modern-day cardiologist understand the diagnostic, prognostic, and therapeutic implications of HCM genetic testing

Reconceptualising temporality in young lives: exploring young people’s current and future livelihoods in AIDS-affected southern Africa

In recent years, anxieties have been expressed that the impacts of southern Africa's AIDS pandemic on young people today will damage their future livelihood prospects. Geographers have been remarkably reluctant to explore young people's future livelihoods, inspired by a concern to view young people as human beings, worthy of study in their own right rather than mere human becomings, of interest only as ‘adults in the making’. Yet there is growing acknowledgement that young people, like older people, are always both ‘being and becoming’. The connections between current and future lives merit much greater attention, both because experiences and actions in childhood and youth undoubtedly shape the futures of individuals and wider society, but also because young people's thoughts and actions are so often geared to the future, and this future orientation shapes their present worlds. This paper reports on research that set out to explore links between the impacts of AIDS and young people's livelihood prospects. Intensive case study research was undertaken, combining participatory methods and life history interviews with young people aged 10–24 in two villages, one in southern Malawi and the other in the mountains of Lesotho. By theorising a temporal dimension to de Haan and Zoomers’ concept of livelihood trajectories, the paper focuses on the ways in which young people respond to both the immediate sustenance requirements of themselves and their households and their need to accrue assets for future livelihoods. Some young people's trajectories appear to be disturbed by the influence of AIDS, but with no systematic patterns. Beyond addressing empirical questions concerning the impacts of AIDS, the paper contributes to our understanding of how livelihoods are produced and to the conceptualisation of youth transitions as produced through the iteration of present and future

A process-dissociation examination of the cognitive processes underlying unconscious thought

Conscious and unconscious thought have been previously found to differentially impact decision-making quality. However, little research has directly measured the processes underlying these modes of thinking. We propose that both thinking modes are characterized by rule-based and intuitive processing. In two experiments, we used the Process Dissociation Procedure to independently measure these cognitive processes. We tested three competing hypotheses: (a) conscious thinking evokes both increased rule-based and decreased intuitive processing compared to unconscious thinking; (b) conscious and unconscious thinking evoke similar levels of intuitive processing but conscious thinking enhances rule-based processing; and (c) conscious and unconscious thinking evoke similar levels of rule-based processing but unconscious thinking enhances intuitive processing. Experiment 1 used base-rate and law-of-large-numbers decision-making problems, whereas Experiment 2 used decision-making problems similar to the "apartment" problem that is often used in unconscious thought studies. In both experiments we found support for hypothesis (b). © 2012 Elsevier Inc

Autonomous Detection of Particles and Tracks in Optical Images

During its initial orbital phase in early 2019, the Origins, Spectral Interpretation, Resource Identification, and Security-Regolith Explorer (OSIRIS-REx) asteroid sample return mission detected small particles apparently emanating from the surface of the near-Earth asteroid (101955) Bennu in optical navigation images. Identification and characterization of the physical and dynamical properties of these objects became a mission priority in terms of both spacecraft safety and scientific investigation. Traditional techniques for particle identification and tracking typically rely on manual inspection and are often time-consuming. The large number of particles associated with the Bennu events and the mission criticality rendered manual inspection techniques infeasible for long-term operational support. In this work, we present techniques for autonomously detecting potential particles in monocular images and providing initial correspondences between observations in sequential images, as implemented for the OSIRIS-REx mission.Comment: 23 pages, 10 figure

Calibration of the instrumental polarization effects of SCExAO-CHARIS’ spectropolarimetric mode

SCExAO at the Subaru telescope is a visible and near-infrared high-contrast imaging instrument employing extreme adaptive optics and coronagraphy. The instrument feeds the near-infrared light (JHK) to the integral field spectrograph CHARIS. Recently, a Wollaston prism was added to CHARIS’ optical path, giving CHARIS a spectropolarimetric capability that is unique among high-contrast imaging instruments. We present a comprehensive and detailed Mueller matrix model describing the instrumental polarization effects of the complete optical path, thus the telescope and instrument, using measurements with the internal source and observations of standard stars. The 22 wavelength bins of CHARIS provide a unique opportunity to investigate in detail the wavelength dependence of the instrumental polarization effects. We find that the image derotator (K-mirror) produces strongly wavelength-dependent crosstalk, in the worst case converting ~95% of the incident linear polarization to circularly polarized light that cannot be measured. We fit the crosstalk of the half-wave plate (HWP) for all wavelengths with a simple two-parameter model of an achromatic HWP consisting of a layer of quartz and a layer of MgF2. While the magnitude of the telescope-induced polarization varies with wavelength, its angle varies solely with the altitude angle of the telescope. We show initial steps toward correcting on-sky data for the instrumental polarization effects, with which we aim to achieve a polarimetric accuracy <0.1% in the degree of linear polarization. Our calibrations of CHARIS’ spectropolarimetric mode enable unique quantitative polarimetric studies of circumstellar disks and planetary and brown dwarf companions

Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides

Bacterial cell-surface display systems coupled with quantitative screening methods offer the potential to expand protein engineering capabilities. To more fully exploit this potential, a unique bacterial surface display scaffold was engineered to display peptides more efficiently from the surface exposed C- and N-termini of a circularly permuted outer membrane protein. Using directed evolution, efficient membrane localization of a circularly permuted OmpX (CPX) display scaffold was rescued, thereby improving the presentation of diverse passenger peptides on the cell surface. Random and targeted mutagenesis directed towards linkers joining the native N- and C-termini of OmpX coupled with screening by FACS yielded an enhanced CPX (eCPX) variant which localized to the outer membrane as efficiently as the non-permuted parent. Interestingly, enhancing substitutions coincided with a C-terminal motif conserved in outer membrane proteins. Surface localization of various passenger peptides and mini-proteins was expedited using eCPX relative to that achieved with the parent scaffold. The new variant also permitted simultaneous display and labeling of distinct peptides on structurally adjacent C- and N-termini, thus enabling display level normalization during library screening and the display of bidentate or dimeric peptides. Consequently, the evolved scaffold, eCPX, expands the range of applications for bacterial display. Finally, this approach provides a route to improve the performance of cell-surface display vectors for protein engineering and design