Microfluidic protein isolation and sample preparation for transmission electron microscopy

The knowledge of atomic structures is essential to understand the mechanics and chemistry of proteins in fundamental research and is often the base for drug development. During the last decades, X-ray crystallography has been the primary method for determining atomic models providing an impressive number of molecular structures. Nevertheless, the technique is limited by the fact that the complexes of interest have to be crystallized. Nuclear magnetic resonance (NMR), which is used as an alternative to solve biomolecules in solution, has the drawback of consuming large amounts of protein, being labour intensive and challenging for large molecules. In recent years, cryogenic electron microscopy (cryo-EM) has evolved as an important tool for protein structure determination. Technical advances in the instrumentation and increased computational power combined with better processing algorithms caused a massive improvement in the resolution of obtained structures. For these achievements Jacques Dubochet, Joachim Frank and Richard Henderson were awarded with a Nobel Prize in 2017. However, sample preparation methods lack behind and did not change a lot. A significant complication is the production of target proteins in sufficient amounts and quality. Although only some thousands to a few million protein particles must be imaged to solve a protein structure, much larger quantities are required to prepare specimens for cryo-EM. Conventional sample preparation methods are very wasteful with proteins and more than 99% of protein is lost during a paper blotting step. Thus, considerable amounts of purified proteins have to be produced using complex and costly procedures usually including several chromatography steps. In this thesis, a novel sample preparation and purification system consuming only minute amounts of biological material is presented. The system allows the purification of proteins and the subsequent preparation of isolated targets for negative stain and cryo-EM. We constructed corresponding hardware and software described in Chapters 1 & 2. The application of the system on biological samples is demonstrated in Chapters 3 & 4. As an example, we purified endogenous human 20S proteasome starting with <1 μL HeLa cytosol and determined it’s 3D structure at a resolution of 3.5Å. In Chapter 5, we show the purification of recombinantly expressed proteins by the use of a novel crosslinker that was developed during the course of this thesis

Two Scenes from Utah\u27s Stratigraphic Record: Neoproterozoic Snowball Earth, Before and After

This research is focused on strata deposited in northern Utah during the Cryogenian Period (850 – 635 Ma) of the Neoproterozoic Era, a period that derives its name from the widespread evidence for multiple, likely global, glacial events during this time, commonly referred to as “Snowball Earth” glaciations. This dissertation includes detailed studies of two Cryogenian successions in northern Utah that bracket potential “Snowball Earth” events: the upper part of the Uinta Mountain Group (deposited prior to the glaciations) and the dolomite member of the Kelly Canyon formation (hypothesized to have formed in the aftermath of a global glaciation that terminated at either 665 or 635 Ma). Both successions contain a lithostratigraphic, geochemical, and biotic record of the Earth’s oceans before and after the largest-magnitude glaciations in the history of our planet. The pre-glacial upper part of the Uinta Mountain Group in the area mapped for this study contains evidence of several (at least three) relatively short periods of ocean anoxia in which ferruginous conditions dominated and euxinia did not occur. There is no evidence that biota (organic-walled microfossil assemblages) were influenced by these brief anoxic events, but evidence from the composite Uinta Mountain Group stratigraphic record does suggest a gradual change in biota similar to that in the Chuar group. It is likely this biotic transition is related to nearshore eutrophication in the oceans, but additional redox geochemical information is needed to fully support this conclusion. The dolomite member of the Kelley Canyon Formation on Antelope Island (post-glacial component of this study) contains idiosyncratic lithologic features thought to be characteristic of 635 Ma deglacial strata, yet its C-isotope values do not lend unequivocal support to this global correlation, and regional correlations and U-Pb zircon ages suggest it is ~30 million years older. These results challenge the popular notion that Neoproterozoic post-glacial cap carbonates can be correlated based upon their lithologic “style,” and they also lend additional support to the possibility of a “Snowball Earth” event at ~665 Ma

Septal myectomy for hypertrophic obstructive cardiomyopathy: coil, boil and the role of rescue surgery

Interventional treatment of hypertrophic obstructive cardiomyopathy has considerably developed and primary surgical approach is nowadays considered for a minority of patients with insufficient relief of obstruction following catheter intervention. We present the history of a patient who underwent alcohol ablation and developed a life-threatening ventricular septal defect consecutively to a large myocardial infarction because of alcohol injection into the LA

Accueil Éducatif Mère-Enfant: le lien d'attachement entre une mère en situation de vulnérabilité et son enfant

Dans le canton de Vaud, les Accueils Éducatifs Mère-Enfant (AEME) reçoivent des femmes enceintes ou élevant seules leur.s enfant.s âgé.s de 0 à 2 ans ayant des difficultés autour du lien mère-enfant. Ces structures, financées par le Service de Protection de la Jeunesse (SPJ), ont pour missions d’accompagner les mères dans la construction du lien avec leur.s enfant.s, de renforcer ou restaurer les compétences maternelles, d’évaluer les conditions de sécurité de l’enfant et d’éviter, dans la mesure du possible, de séparer le bébé de sa mère

A spatial mixed Poisson framework for combination of excess-of-loss and proportional reinsurance contracts

In this paper a purely theoretical reinsurance model is presented, where the reinsurance contract is assumed to be simultaneously of an excess-of-loss and of a proportional type. The stochastic structure of the set of pairs (claim’s arrival time, claim’s size) is described by a Spatial Mixed Poisson Process. By using an invariance property of the Spatial Mixed Poisson Processes, we estimate the amount that the ceding company obtains in a fixed time interval in force of the reinsurance contract