Development of an in vitro system for continuous directed evolution of high affinity protein ligands

Die gerichtete Evolution ermöglicht die Modifikation von Proteinen und Nukleinsäuren nach den drei Prinzipien von Darwin - Mutation, Selektion und Replikation. Bisherige Methoden sind durch ihre diskontinuierliche Form und ihre Variantenanzahl limitiert. Ziel dieser Arbeit war die Etablierung eines Verfahrens zur kontinuierlichen, gerichteten Evolution hochaffiner Protein-Liganden in vitro. Dabei wurde erstmals die Kombination einer fehlerbehafteten DNA-Amplifikation (NASBA) mit einem in vitro Proteinsynthesesystem gezeigt. Verschiedene Parameter der NASBA wurden hinsichtlich einer effizienteren Amplifikation von DNA-Templates optimiert. Mutationen, basierend auf der fehlerbehafteten Aktivität von T7 RNA Polymerase und MMuLV RT, wurden analysiert und die Mutationsraten mit Hilfe des seriellen Transfer-Prinzips gesteigert. Für den Selektionsschritt, basierend auf der Affinität zweier Interaktionspartner, wurde die Expression und Aufreinigung von T7 RNA Polymerase-Fusionsproteinen optimiert. Die Fusion der zu evolvierenden Bindepartner an die MMuLV RT zeigte einen vernachlässigbaren Effekt auf die Reverse Transkriptase-Aktivität. Für die Kombination von NASBA und in vitro Proteinexpression wurde das PURE System, das im Gegensatz zu E.coli Lysaten die DNA-Amplifikation nicht inhibierte, verwendet. Nach Optimierung der Nukleotid- und Magnesiumionenkonzentration konnte erstmals eine funktionelle Kombination von NASBA und in vitro Proteinsynthese in einem Reaktionsansatz stattfinden. Weiterhin wurde gezeigt, dass das NASBA-Produkt als Template für die Proteinexpression fungieren kann und eine endogen exprimierte MMuLV RT ausreichend Aktivität besitzt, um die NASBA anzutreiben. Versuche mit einem Test-Interaktionspaar ergaben, dass der Selektionsschritt innerhalb des Systems weiterer Optimierung bedarf. Damit wurden grundlegende Voraussetzungen für ein System zur kontinuierlichen, gerichteten Evolution hochaffiner Protein-Liganden geschaffen.Directed evolution based on the three principles of mutation, selection and replication has become a popular strategy to modify proteins and nucleic acids. But today’s methods for directed evolution are still limited in their variant diversity and discontinuous in their nature. Aim of this work was to establish an in vitro system for continuous directed evolution of high affinity protein ligands. Thereby, an erroneous DNA amplification method was combined with an in vitro protein synthesis system for the first time. NASBA, an isothermal method to amplify short RNA molecules, was optimised regarding incubation temperature, buffer conditions, enzymes and primers to achieve efficient amplification of DNA templates. Mutations due to the error-prone activities of T7 RNA polymerase and MMuLV RT were analysed and mutation rates could be increased by applying the serial transfer principle. Selection should rely on the affinity of two interacting partners. Expression and purification parameters of T7 RNA polymerase fusion proteins were optimized. The fusion of MMuLV RT to binding partners only hardly affected reverse transcriptase activity. To combine NASBA and in vitro protein expression the PURE System was chosen, which in contrary to E. coli lysates caused no inhibition of DNA amplification during NASBA. The concentrations of nucleotides and magnesium ions turned out to be highly critical and were readjusted to facilitate a functional combination of NASBA and in vitro protein synthesis within one reaction. It was shown that NASBA products can function as templates for protein expression and that endogenously expressed MMuLV RT exhibits enough activity to drive NASBA amplification. First trials with an interacting couple revealed that both - NASBA and in vitro protein synthesis - operate simultaneously, but the selection step needs to be further optimised. In summary, fundamental preconditions for a continuous directed evolution system were established within this work

Investigations of Biomass Pretreatment and Submerged Fixed-bed Fermentation

To improve the MixAlco process and biomass pretreatment, five studies were conducted. Three studies related to fermentation, whereas the other two investigated the effectiveness of shock tube pretreatment (STP) coupled with oxidative lime pretreatment (OLP). In the first study, the constant-selectivity assumption used in the continuum particle distribution model (CPDM) was determined to be invalid. During a 32-day batch fermentation, selectivity increased from 0.10 to 0.40 g acid/g non-acid volatile solid (NAVS) digested. Future revisions to CPDM should incorporate a non-constant selectivity term. In the second study, a revised procedure was developed to provide a more accurate determination of moisture content. Conventional drying at 105 degrees C allowed product acids to vaporize with water, which introduced errors. Using the revised procedure, calcium hydroxide or sodium hydroxide was added to samples at a concentration of 0.01 g base/g sample, which retained acids in the sample. The mass of additional retained material closely matched that of the additional retained acid. Three related studies involving biomass pretreatment were performed. In the first, recommended parameters for pretreating sugarcane bagasse with OLP and STP were determined. Recommended OLP parameters were 130 degrees C, 6.9-bar O2, and 2-h duration. The effects of solids concentration, liquid fill volume, particle size, type of shotgun shell, number of shocks, and pretreatment order were investigated. Liquid fill volume, particle size, type of shotgun shell, and pretreatment order were significant variables, whereas solids concentration and number of shocks were not. Recommended OLP parameters were used as a basis for an additional experiment. To simulate industrial-scale pile fermentation, fixed-bed batch fermentation of OLP + STP sugarcane bagasse was performed in 1-L PVC fermentors. Rubber mulch was used as a structural support material to prevent filter plugging, which had been reported in previous work. After 42 d, acid concentration reached 8 g/L with yield approximately 0.1 g acid/g NAVS fed. Poor fermentation performance was caused by short solid-liquid contact time and poor pH control. A third biomass pretreatment experiment investigated the potential of pretreated corn stover as a potential ruminant feed. Five samples (raw, OLP, STP, OLP + STP, and STP + OLP) were analyzed for composition and in vitro digestibility. STP followed by OLP increased neutral detergent fiber (NDF) digestibility from 49.3 to 79.0 g NDF digested/100 g NDF fed. On an organic matter basis, STP + OLP corn stover plus water-soluble extractives had a total digestible nutrients (TDN) of 74.9, nearly reaching corn grain at 88.1

Structural and chemical characterization of the back contact region in high efficiency CdTe solar cells

Cadmium telluride (CdTe) is the leading commercialized thin-film photovoltaic technology. Copper is commonly used in back contacts to obtain high efficiency, but has also been implicated as a harmful factor for device stability. T hus it is critical to understand its composition and distribution within complete devices. In this work the composition and structure of the back contact region was examined in high efficiency devices (-16%) contacted using a ZnTe:Cu buffer layer followed by gold metallization. T he microstructure was examined in the asdeposited state and after rapid thermal processing (RTP) using high resolution transmission electron microscopy and EDX chemical mapping. After RTP the ZnTe exhibits a bilayer structure with polycrystalline, twinned grains adjacent to Au and an amorphous region adjacent to CdTe characterized by extensive Cd-Zn interdiffusion. T he copper that is co-deposited uniformly within ZnTe is found to segregate dramatically after RTP activation, either collecting near the ZnTe/Au interface or forming CUxTe clusters in CdTe at defects or grain boundaries near the interface with ZnTe. Chlorine, present throughout CdTe and concentrated at grain boundaries, does not penetrate significantly into the back contact region during RTP activation

Congenital Hypogonadotropic Hypogonadism Due to GNRH Receptor Mutations in Three Brothers Reveal Sites Affecting Conformation and Coupling

Congenital hypogonadotropic hypogonadism (CHH) is characterized by low gonadotropins and failure to progress normally through puberty. Mutations in the gene encoding the GnRH receptor (GNRHR1) result in CHH when present as compound heterozygous or homozygous inactivating mutations. This study identifies and characterizes the properties of two novel GNRHR1 mutations in a family in which three brothers display normosmic CHH while their sister was unaffected. Molecular analysis in the proband and the affected brothers revealed two novel non-synonymous missense GNRHR1 mutations, present in a compound heterozygous state, whereas their unaffected parents possessed only one inactivating mutation, demonstrating the autosomal recessive transmission in this kindred and excluding X-linked inheritance equivocally suggested by the initial pedigree analysis. The first mutation at c.845 C>G introduces an Arg substitution for the conserved Pro 282 in transmembrane domain (TMD) 6. The Pro282Arg mutant is unable to bind radiolabeled GnRH analogue. As this conserved residue is important in receptor conformation, it is likely that the mutation perturbs the binding pocket and affects trafficking to the cell surface. The second mutation at c.968 A>G introduces a Cys substitution for Tyr 323 in the functionally crucial N/DPxxY motif in TMD 7. The Tyr323Cys mutant has an increased GnRH binding affinity but reduced receptor expression at the plasma membrane and impaired G protein-coupling. Inositol phosphate accumulation assays demonstrated absent and impaired Gαq/11 signal transduction by Pro282Arg and Tyr323Cys mutants, respectively. Pretreatment with the membrane permeant GnRHR antagonist NBI-42902, which rescues cell surface expression of many GNRHR1 mutants, significantly increased the levels of radioligand binding and intracellular signaling of the Tyr323Cys mutant but not Pro282Arg. Immunocytochemistry confirmed that both mutants are present on the cell membrane albeit at low levels. Together these molecular deficiencies of the two novel GNRHR1 mutations lead to the CHH phenotype when present as a compound heterozygote

Note d'exégèse : une nouvelle conjecture à propos , du psaume 4, verset 7 b

La plupart des spécialistes reconnaissent que le troisième caractère du verbe nsh représente une consonne corrompue. L'auteur aborde cette corruption du point de vue de la phonétique générale. Il voit dans le h le dernier vestige d'une consonne accommodée à la première consonne du mot suivant, un 'ayin. L'articulation du 'ayin, préparé pendant la prononciation de la dernière consonne du verbe l'aurait changé en h. Seul le verbe nsk donne une phrase satisfaisante : « Répands sur nous la lumière de ta face. » Le changement du kaf (palatal) en he (guttural) s'explique, de fait, par accommodation au 'ayin (guttural) suivant.Meysing Jacques. Note d'exégèse : une nouvelle conjecture à propos , du psaume 4, verset 7 b. In: Revue des Sciences Religieuses, tome 40, fascicule 2, 1966. pp. 154-157

Contribution à l'étude des généalogies bibliques. Technique de la composition des chronologies babyloniennes du Déluge

On cherche à déterminer, autant que possible, les moyens et méthodes utilisés par les chronologies babyloniennes du déluge, pour éclairer le genre littéraire des généalogies bibliques.Meysing Jacques. Contribution à l'étude des généalogies bibliques. Technique de la composition des chronologies babyloniennes du Déluge. In: Revue des Sciences Religieuses, tome 39, fascicule 3, 1965. pp. 209-229

La chronographie juive à l'époque gréco-romaine

La croyance en un rajeunissement périodique du vieil univers était liée à la pensée astrologique. On calculait donc les révolutions célestes et on se hasardait à fixer la durée de la Grande Année. La chronographie apocalyptique ambitionnait d'adapter les données historiques au cadre de la chronologie céleste, en vue de la prédiction de la fin des temps. La division de l'histoire en périodes en était la conséquence. A partir de l'an 70 de notre ère, on constate une contraction progressive des périodes successives de la chronologie de la Bible hébraïque. Ce mouvement d'abrègement s'arrête dans la première moitié du IIème siècle. La première phase de cette contraction progressive s'explique par le phénomène parallèle de l'eschatologie différée La phase ultérieure du même mouvement signifie qu'on avait abandonné l'attente immédiate de l'eschaton en faveur de l'idée d'une répétition cyclique de l'histoire.Meysing Jacques. La chronographie juive à l'époque gréco-romaine. In: Revue des Sciences Religieuses, tome 41, fascicule 4, 1967. pp. 289-304

Introduction à la numérologie biblique. - Le diagramme Sator-Arepo

Dans la religion astrale passé et avenir se déroulaient selon un rythme cosmique, symbolisé par la Roue du Temps. La divination et la chronographie s'inspiraient d'elle. Le carré SATOR fut composé d'après le modèle du diagramme horoscopique. Ensuite, des chronologies juives et non-juives, ainsi que des listes de nombres de versets du Livre Sacré sont marquées par une numérologie cosmique. A l'arrière fond de cette numérologie serait, de nouveau, la Roue du Temps. L'auteur du carré SATOR était au courant de la cryptographie grecque. Des chronologistes juifs et non-juifs utilisaient la même technique pour la transformation d'un diagramme de nombres en une série linéaire. Une telle chronologie est donc un diagramme de nombres cryptographie.Meysing Jacques. Introduction à la numérologie biblique. - Le diagramme Sator-Arepo. In: Revue des Sciences Religieuses, tome 40, fascicule 4, 1966. pp. 321-352