Novel Approaches to the Production & Analysis of Biological Nanomaterials for Serial- Femtosecond X-ray Crystallography

Tremendous effort has recently been put into the development of novel methods and techniques in the realm of serial-femtosecond x-ray crystallography (SFX) and x-ray free-electron lasers (XFEL). This thesis strives to contribute to current research approaches. This is done by focusing on the exploration of methods that can accommodate the increasing demand for nano-sized samples in serial data collection. The presented research focuses on the investigation of the peroxisomal system of the budding yeast speciesH. polymorpha and S. cerevisiae in particular. Both are potential in vivo factories and carriers of crystalline material from heterologously expressed proteins. The outstanding features of the yeast eukaryotic systems, their size, robustness and versatility indicate optimal conditions for such efforts. Using the example of naturally-occuring peroxisomal AOX crystals in the yeast H. polymorpha, it is shown that meaningful diffraction data can be obtained at modern XFEL sources via the injection of whole yeast cells intothe x-ray beam. The yeast systems demonstrate robustness under the applied harsh experimental conditions (injection via a GDVN nozzle). Thus, proof is given that yeast cells are a suitable envelope to protect fragile, nano-sizedprotein crystals. To build up on the idea of creating a productive in vivo ’crystal factory’, it is further shown how to characterize in vivo crystals by means of synchrotron powder diffraction and a Williamson-Hall analysis. These methods enable to quantify crystal domain sizes and strain and can beused in future attempts to evaluate and optimize crystal quality ahead of FEL experiments. It has also been demonstrated how to obtain superoxide dismutase 1 (SOD1)amyloid-fibers, so as to provide suitable samples for serial data collection from non-crystalline material. In relation to results from dynamic light scattering (DLS), electron microscopy (EM) and fluorescence-binding assays (ThT as-say), possible mechanisms are discussed by which protein fibers are formed. Moreover, it has been attempted to utilize the optimized fiber sample for serial femtosecond fiber diffraction at a XFEL source. In order to investigate the accumulation and possible nucleation/crystallizationof heterologously expressed proteins in the peroxisomal environment, a new approach has been developed. It relies on the combination of intraperoxisomal, fluorescent probe molecules and the observation of their steady-state fluorescence anisotropy. A theoretical model is developed to which experimental data can be compared in terms of fluorescence anisotropy and energy transfer. Using the example of the protein EGFP it is shown that yeast cells require further genetic engineering to corroborate the hypothesis of an ever-increasing intraperoxisomal protein concentration. Consequently, the occurrence of AOX crystals seems also not to be induced by extremely high protein concentrations.Proceeding from the established anisotropy method, a gedankenexperiment is conducted to demonstrate the possibility to distinguish protein clustersfrom crystals in case the influence of the FRET orientation factor $κ^2 is being considered in measurements of fluorescence anisotropy and homoFRET

Erosion hydrique et restauration des sols. 15 ans de travaux menes par les organismes de recherche et les societes de developpement francais

CNRS RP 185 (1499) / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEFRFranc

In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. The observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outline

Paysage ou territoires ? Deprise agricole et interpretation de l'espace

Available at INIST (FR), Document Supply Service, under shelf-number : RP 185 (2661) / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEFRFranc

Etude comparee des formes metalliques du plomb et du cadmium en deux zones cotieres de transfert. (Port de Dunkerque; estuaire de la Canche)

SIGLECNRS RP 185 (1768) / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Perception de l'eau diffuse et enjeux d'utilisation d'un espace agraire. Representations, pratiques et strategies de gestion de l'espace dans un pays mouille

SIGLECNRS RP 185 (2100) / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Via placement optimization for a group of wires

Most PCB design CAD systems offer a limited number of “patterns” for the via placement on a bus (group of wires) which would be either a single- or a double-row placement. This article demonstrates the incorrectness of such limitations, because in this case the mounting space is used not in an optimal way. The paper presents the optimum solution for a certain type of problems on via placement when changing the layer of a bus. The solution suggests a regular (periodic) arrangement, but with a multi-row placement. The calculation of the parameters for optimal placement is narrowed, in general, to finding the number of via rows with which the area of a topological fragment is minimal

Pouvoir syndical et revendications ouvrieres dans les entreprises en reconversion : Republique Tcheque, Pologne et Hongrie

Available from INIST (FR), Document Supply Service, under shelf-number : AR 16530 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEMinistere de la Recherche et de l'Espace (MRE), 75 - Paris (France)FRFranc