L'alpha-1 antitrypsine (A1AT) est un inhibiteur de sérine protéase sécrété principalement par le foie et libéré dans la circulation où sa concentration physiologique est de 1,5 à 3,5 g/L. La principale fonction de l'A1AT est d'inhiber l'activité de l'élastase des neutrophiles (NE) afin de maintenir l'équilibre protéase/anti-protéase dans les poumons. Son déficit (A1ATD) touche plus de 3,4 millions d'individus dans le monde chez qui l'élastase des neutrophiles décompose l'élastine, provoquant ainsi une diminution de l'élasticité du poumon ainsi qu'une dégradation de son tissu conjonctif. En conséquence, l'A1ATD entraîne des troubles respiratoires tels que l'emphysème ou la maladie pulmonaire obstructive chronique et ceux qui en sont atteints nécessitent des injections fréquentes d'A1AT purifiée à partir du sang d'un donneur. Cependant, l'A1AT plasmatique est hétérogène dans son état de glycosylation et sa qualité varie d'un lot à l'autre. De plus, il y a un risque, même très faible, de transmission d'agents pathogènes avec l'administration d'A1AT purifiée par plasma. Par conséquent, il existe un besoin pour une version recombinante. La glycoprotéine mature possède trois sites de N-glycosylation comprenant principalement des structures de type complexe bi-antennaires afucosylées et α-2,6-di-sialylées, A2G2S2 (6,6). Bien que la glycosylation ne soit pas essentielle à l'activité inhibitrice de l'A1AT, il a été démontré qu'elle a un impact significatif sur sa demi-vie in vivo. Notamment, l'acide sialique, un monosaccharide terminal chargé négativement présent sur les N-glycanes, aide à prolonger la demi-vie de l'A1AT dans le sérum en empêchant l'interaction entre l'avant-dernier galactose (Gal) du N-glycane et les récepteurs hépatiques des asialoglycoprotéines (ASGPRs), composés de deux sous-unités appelées lectines hépatiques (HL) 1 et 2, qui se lient aux glycoprotéines asialylées contenant un Gal terminal et conduisent à leur dégradation. Par conséquent, il est important de produire A1AT dans un système d'expression qui peut effectuer les modifications post-traductionnelles (PTM) appropriées à des fins thérapeutiques. Jusqu'à présent, la production d'A1AT recombinante (rA1AT) a été tentée dans différents systèmes d'expression cellulaire avec un succès limité. Malgré la disponibilité de diverses lignées cellulaires, les cellules ovariennes de hamster chinois (CHO) ont été largement utilisées pour la production de glycoprotéines thérapeutiques car ces cellules sont compatibles avec des stratégies de glyco-ingénierie pour produire des glycoprotéines recombinantes composées de glycanes de type humain. Cependant, ces cellules synthétisent des N-glycanes de type complexe comprenant de la fucosylation centrale et de l'acide sialique lié en α-2,3. Par conséquent, dans ce projet, l'objectif était de développer une version recombinante d'A1AT avec un profil de glycosylation humaine exprimée en cellules CHO modifiées et qui se prête à des utilisations thérapeutiques. À cette fin, dans notre étude, nous avons d'abord empêché l'α-2,3 sialylation ainsi que la fucosylation centrale en éliminant les gènes responsables via la technologie CRISPR/Cas9, suivie de la surexpression de l'α-2,6‐sialyltransférase humaine à l'aide d'un système d'expression inductible au cumate. Nous avons ensuite montré la supériorité du promoteur inductible CR5 pour l’expression de A1AT par rapport à cinq promoteurs constitutifs forts couramment utilisés dans l'industrie. En utilisant le promoteur CR5, nous avons généré des populations de CHO stables modifiées par glyco-ingénierie produisant plus de 2,1 g/L pour la forme native et 2,8 g/L pour la version mutée d'A1AT avec des N-glycanes analogues au produit clinique dérivé du plasma, la Prolastin-C. L'effet bénéfique de la supplémentation en N‐acétylmannosamine du milieu de culture cellulaire sur la glycosylation de l'A1AT a également été démontré. Enfin, nous avons montré que l'activité anti‐élastase des rA1ATs est comparable à celle de la Prolastin-C, et que la substitution des résidus méthionines critiques par des valines rendait A1AT significativement plus résistante à l'oxydation. Nous avons ensuite étudié l'impact de la glycosylation d'A1AT sur son interaction avec les orthologues d'ASGPR. Pour cela, nous avons initialement utilisé un test d'internalisation cellulaire basé sur la lignée cellulaire hépatique humaine HepG2 connue pour exprimer les ASGPRs à sa surface et avons examiné leur interaction avec les rA1ATs possédant divers profils de glycosylation. Comme le test d'internalisation basé sur les cellules HepG2 a démontré un faible rapport signal sur bruit (SNR) ainsi qu'un niveau élevé de signal de fond d'internalisation, nous avons cherché à développer un nouveau test basé sur des cellules CHO surexprimant des orthologues ASGPR recombinants. Alors que la sous-unité HL-1 humaine seule était suffisante pour lier et internaliser l'A1AT asialylée, les sous-unités HL-1 et HL-2 étaient nécessaires pour former des récepteurs fonctionnels et ayant une forte affinité pour les ASGPR de rat et de souris. Afin d'améliorer le SNR de notre test cellulaire d'internalisation, le tri cellulaire activé par fluorescence (FACS) a été utilisé pour enrichir les populations de cellules CHO pour celles exprimant des niveaux élevés d'orthologues ASGPR. Enfin, en utilisant des structures de glycanes remodelés par voie enzymatique de Prolastin-C, nous n'avons observé aucune internalisation lorsque les glycanes sont terminés avec α-2,6-Neu5Ac ni α-2,8-Neu5Ac-α-2,6-Neu5Ac par l’ASGPR de l'humain, du rat et de la souris. D'autre part, l'absorption de Prolastin-C portant des glycanes bi-antennaires avec une branche terminée par de l'acide sialique α-2,3 et l'autre par du galactose terminal, par l'ASGPR de souris a été statistiquement plus élevée que celle de l'humain et du rat. En somme, l'A1AT recombinante résistante à l'oxydation décrite dans ce projet pourrait représenter un meilleur médicament biothérapeutique tout en offrant une alternative sûre et plus stable pour la thérapie d'augmentation. Nous avons également contribué à une meilleure compréhension de l'impact de la sialylation de l'A1AT sur son internalisation cellulaire par les orthologues ASGPR. / Alpha-1 antitrypsin (A1AT) is a serine protease inhibitor secreted primarily by the liver, and released in the circulation where its physiological concentration is 1.5-3.5 g/L. The main physiological function of A1AT is to inhibit the activity of neutrophil elastase (NE) to maintain the protease/anti-protease balance in the lung. The A1AT deficiency (A1ATD) is affecting more than 3.4 million individuals worldwide where neutrophil elastase breaks down elastin, thereby causing a decrease in the elasticity of the lung as well as a degradation of its connective tissue. As a result, A1ATD leads to respiratory disorders such as emphysema or chronic obstructive pulmonary disease. Treatment of this health condition requires frequent injections of A1AT purified from donor blood. However, plasma A1AT is heterogeneous in its glycosylation state and its quality varies from batch to batch. Moreover, there is a risk, however very low, of pathogen transmittance with plasma-purified A1AT administration. Therefore, there is a need for recombinant version. The mature glycoprotein has three N-glycosylation sites possessing mostly afucosylated, α-2,6-di-sialylated bi-antennary complex-type structures, A2G2S2 (6,6). Though glycosylation is not essential for A1AT's inhibitory activity, it has been shown to have a significant impact on its in vivo half-life. Notably, sialic acid, a terminal negatively charged monosaccharide present on N-glycans, helps to prolong the half-life of A1AT in serum by preventing the interaction between the penultimate galactose (Gal) of the N-glycan and the hepatic asialoglycoprotein receptors (ASGPRs), composed of two subunits termed hepatic lectin (HL) 1 and 2, which bind to asialylated glycoproteins containing terminal Gal and lead to their degradation. To this extend, it is important to produce A1AT in an expression system that can carry out the appropriate post-translational modifications (PTMs) for therapeutic purposes. Thus far, the production of recombinant A1AT (rA1AT) has been attempted in different cell expression systems with limited success. Despite the availability of various cell lines, Chinese hamster ovary (CHO) cells have been widely used to produce therapeutic glycoproteins as these cells can tolerate glycoengineering strategies to produce recombinant glycoproteins with human-like glycans. However, these cells synthesize complex-type N-glycans with core-fucosylation along with α-2,3-linked sialic acid. Therefore, in this research project, the aim was to develop a recombinant version of A1AT with human glycosylation pattern expressed in genetically engineered CHO cells that would be amenable to therapeutic uses. To this end, in our study, we first prevented α-2,3 sialylation as well as core-fucosylation by eliminating the corresponding genes via CRISPR/Cas9 technology, followed by overexpressed human α-2,6‐sialyltransferase using a cumate‐inducible CHO expression system. We then showed superiority of the CR5 inducible promoter compared to five strong constitutive promoters commonly used in the industry. Using the CR5 promoter, we generated glycoengineered stable CHO pools producing over 2.1 g/L of the wild-type and 2.8 g/L of the mutein forms of A1AT, with N‐glycans analogous to the plasma‐derived clinical product, Prolastin-C. The effect of N‐acetylmannosamine supplementation to the cell culture media on the A1AT glycosylation was also demonstrated. Finally, we showed that the anti‐elastase activity of rA1ATs is comparable to that of Prolastin-C, and that substitution of critical methionine residues with valines rendered A1AT significantly more resistant to oxidation. We then studied the impact of A1AT glycosylation on its interaction with ASGPR orthologs. For this, we initially used a cell-based internalization assay based on the human HepG2 hepatic cell line known to express ASGPRs at its surface and examined their interaction with rA1ATs possessing various glycosylation profiles. As HepG2 cell-based internalization assay demonstrated poor signal-to-noise ratio (SNR) as well as high level of background internalization signal, we then aimed at developing a new assay based on CHO cells overexpressing recombinant ASGPRs orthologs. While human HL-1 subunit alone was sufficient to bind and internalize asialylated A1AT, both HL-1 and HL-2 subunits were required to form functional and high affinity receptors for the rat and mouse ASGPRs. To enhance SNR of our cell-based uptake assay, fluorescence-activated cell sorting (FACS) was used to enrich the CHO pools for cells expressing high levels of ASGPR orthologs. Finally, using enzymatically remodelled glycan structures of Prolastin-C, we observed no uptake when glycans are terminated with α-2,6-Neu5Ac nor α-2,8-Neu5Ac-α-2,6-Neu5Ac by human, rat, and mouse ASGPR orthologs. On the other hand, the uptake of Prolastin-C bearing bi-antennary glycans with one branch terminated with α-2,3 sialic acid and the other with terminal galactose, by mouse ASGPR was observed to be statistically higher than that by human and rat ASGPR orthologs. Collectively, the oxidation-resistant recombinant A1AT described in this project could represent a viable biobetter drug while offering a safe and more stable alternative for augmentation therapy. We also contributed a better understanding of the impact of A1AT sialylation on its cellular uptake by ASGPR orthologs.

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Welding is used extensively in aerospace, automotive, chemical, manufacturing, electronic and power-generation industries. Thermally-induced residual stresses due to welding can significantly impair the performance and reliability of welded structures. Numerical simulation of weld pool dynamics is important as experimental measurements of velocities and temperature profiles are difficult due to the small size of the weld pool and the presence of the arc. From a structural integrity perspective of welded structures, it is necessary to have an accurate spatial and temporal thermal distribution in the welded structure before stress analysis is performed. Existing research on weld pool dynamics simulation has ignored the effect of fluid flow in the weld pool on the temperature field of the welded joint. Previous research has established that the weld pool depth/width (D/W) ratio and Heat Affected Zone (HAZ) are significantly altered by the weld pool dynamics. Hence, for a more accurate estimation of the thermally-induced stresses it is desired to incorporate the weld pool dynamics into the analysis. Moreover, the effects of microstructure evolution in the HAZ on the mechanical behavior of the structure need to be included in the analysis for better mechanical response prediction. In this study, a three-dimensional model for the thermo-mechanical analysis of Gas Tungsten Arc (GTA) welding of thin stainless steel butt-joint plates has been developed. The model incorporates the effects of thermal energy redistribution through weld pool dynamics into the structural behavior calculations. Through material modeling the effects of microstructure change/phase transformation are indirectly included in the model. The developed weld pool dynamics model includes the effects of current, arc length, and electrode angle on the heat flux and current density distributions. All the major weld pool driving forces are included, namely surface tension gradient, plasma drag force, electromagnetic force, and buoyancy. The weld D/W predictions are validated with experimental results. They agree well. The effects of welding parameters (like welding speed, current, arc length, etc.) on the weld D/W ratio are documented. The workpiece deformation and stress distributions are also highlighted. The transverse and longitudinal residual stress distribution plots across the weld bead and their variations with welding speed and current are also provided. The mathematical framework developed here serves as a robust tool for better prediction of weld D/W ratio and thermally-induced stress evolution and distribution in a welded structure by coupling the different fields in a welding process. / Ph. D.

<p dir="ltr">Grid convergence and scaling analyses have not been done rigorously for practical large-eddy simulations (LES). The challenge arises from the fact that there are two grid-related length scales: grid size and LES filter width. It causes the numerical and model errors in LES to be inherently coupled, making the convergence of either error difficult to analyze. This study works to overcome the challenge by developing scaling laws that can be used to guide the convergence analysis of errors in LES. Three different convergence cases are considered, and their respective scaling laws are developed by varying the ratio between grid size and filter width. A pool fire is adopted as a test case for the convergence analysis of LES. Qualitative and quantitative assessments of the LES results are made first to ensure reliable numerical solutions. In the subsequent scaling analysis, it is found that the results are consistent with their respective scaling laws. The results provide strong support to the developed scaling laws. The work is significant as it proposes a rigorous way to guide convergence analysis of LES errors. In a world where LES already has a wide range of applicability and is still becoming more prominent, it is imperative to have a thorough understanding of how it works including its convergence and scaling laws with respect to the change of grid size and filter width.</p>

Title: The Analysis of Selected Swimming Pools in Prague Objectives: The aim of my thesis is to create an analysis of selected swimming pools and to compare them to each others. Furthermore, the thesis should show the threats and opportunities of the selected swimming pools. Methods: The case analysis was chosen as the main research method. In the preparation of this analysis other methods were applied, such as intentional selection, document analysis and semi-structured interview. Based on the results the SWOT analysis was developed. Through this method the strengths, weaknesses, opportunities and threats of the selected swimming pools are shown. The final comparison of all the swimming pools was made using all the previously mentioned methods. Results: The analysis shows that we can label all the selected swimming pools in Prague as functional swimming pools. Functional swimming pools do not lose money, nor do they profit. However, it was proved that none of the chosen swimming pools would be able to function without getting some form of grants. The location of a swimming pool does not influence the number of visitors coming there. Every location has its advantages and disadvantages. The selected swimming pools should try to make their web sites more interesting and easily accessible to visitors,...

The present PhD thesis describes the synthesis and characterization of novel planarchiral 1,2-P,O-ferrocenes and their application in the Pd-catalyzed Suzuki-Miyaura reaction. It was especially focused on the development of a new synthetic pathway to this type of substitution pattern by applying the anionic phospho-Fries rearrangement in ferrocene chemistry. Starting from hydroxy ferrocene, a high diversity of Fc–O–P- type (Fc = (n5-C5H5)(n5-C5H4)) compounds were synthesized, whereby the electronic properties of the phosphorus fragments were varied. The anionic phospho-Fries rearrangement successfully occurred subsequent to an ortho-lithiation with a non-nucleophilic base giving the 1,2-P,O-ferrocenes in up to quantitative yields. The usage of chiral pool-based alcohols for the synthesis of chiral ferrocenyl phosphates allowed a diastereoselective proceeding, giving single isomers in up to 95% de. Temperature-dependent investigations of mixed ferrocenyl-, phenyl- and Nheterocyclicphosphates revealed a limitation of ferrocenyl-based rearrangements per reaction step, contrary to phenyls. 1,2-P,O-ferrocenyl phosphonates could successfully be converted into phosphines by applying Stelzer P,C cross-coupling reactions on ferrocenes for the first time. Their usage as ligands for C,C cross-coupling reactions was confirmed by the synthesis of sterically hindered biaryls in high yields at 70°C with a low catalyst loading of 1 mol-% Pd. Functionalization of the 1,2-P,O -structural motif could be achieved by applying nucleophilic aromatic substitution reactions (SNAr) as an alternative pathway for the synthesis of ferrocenyl aryl ethers. Subsequent to a Fries rearrangement sterically-demanding 1,3-di-orthosubstituted aryloxy ferrocenes could be obtained. Multi SNAr reactions of hydroxyferrocenes at polyfluorinated arenes gave up to pentaferrocenyl-functionalized aryl ethers, whose electrochemical properties were investigated. The reaction of CH2-enlarged ferrocenylmethanols gave α-ferrocenylcarbenium ions instead of phosphates, while treating them with chlorophosphates. Enantiopure 2-P(S)Ph2-substituted derivatives of these ions underwent a subsequent intermolecular “S2−“ migration, resulting in thioethers, for example (Sp,Sp)-(2-(P(S)Ph2)FcCH2)2S, in a unique mechanism. Instead, the presence of electronrich arenes gave electrophilic aromatic substituted benzenes bearing chiral ferrocenylmethyl backbones. These type of ligands gave biaryls with up to 26 % ee. / Die vorliegende Arbeit beschäftigt sich mit der Synthese und Charakterisierung von neuartigen planar-chiralen 1,2-P,O-ferrocenen sowie deren Anwendung in der Pd-katalysierten Suzuki-Miyaura Reaktion. Dabei lag der Focus in der Anwendung anionischer phospho-Fries Verschiebungen auf Ferrocene um dieses spezielle Substitutionsmuster erhalten zu können. Ausgehend von Hydroxyferrocenen wurde so eine Vielzahl neuer Verbindungen des Typs Fc–O–P (Fc = (n5-C5H5)(n5-C5H4)) synthetisiert, wobei die elektronischen Eigenschaften des Phosphor-tragenden Fragmentes variiert wurden. Die anionische phospho-Fries Verschiebung erfolgte im Anschluss einer ortho-Lithiierung mit einer nicht-nucleophilen Base, was 1,2-P,O-Ferrocene nahezu quantitative ergab. Durch die Verwendung von chiral-pool basierten Alkoholen für die Synthese chiraler Ferrocenylphosphate konnte eine diastereoselective 1,3-O->C Umlagerung mit Isomerenreinheiten von bis zu 95 % erreicht werden. Untersuchungen von gemischten Ferrocenyl-, Phenyl- und N-heterocyclischen Phosphaten bei verschiedenen Temperaturen zeigte eine Limitierung Ferrocenyl-basierter Umlagerungen pro Reaktionsschritt im Gegensatz zu denen von Phenylen. Die erhaltenen 1,2-P,O-Ferrocenylphosphate wurden erstmals mittels Stelzer P,C Kreuz-Kupplungsreaktionen erfolgreich in Phosphane umgesetzt. Deren Verwendung als Ligand in C,C Kreuz-Kupplungsreaktionen wurde am Beispiel der Synthese räumlich anspruchsvoller Biaryle in hohen Ausbeuten bei nur 70°C und niedrigen Katalysatorbeladungen von 1 mol-% Pd demonstriert. Die Funktionalisierung des 1,2-P,O-Strukturmotifes konnte durch die Anwendung von nucleophilen aromatischen Substitutionsreaktionen erreicht werden, die einen alternativen Zugang zu Ferrocenylarylethern bietet. Im Anschluss an eine Fries Umlagerung konnten so sterisch anspruchsvolle 1,3-di-ortho-substituierte Ferrocenylarylether erhalten werden. Durch mehrfache SNAr Reaktionen von Hydroxyferrocenen an polyfluorierten Aromaten wurden bis zu fünf-fach substituierte Ether erhalten, deren elektrochemischen Eigenschaften untersucht wurden. Die um eine CH2-Einheit verlängerten Ferrocenylmethanole gaben bei Reaktion mit Chlorophosphaten alpha Ferrocenylcarbeniumionen anstelle der gewünschten Phosphate. Enantiomerenreine 2-P(S)Ph2-substituierte Derivate dieser Ionen durchliefen anschließend einen intermolekularen ”S2–“ Transfer in einem einzigartigen Mechanismus, was bspw. in Thioethern des Typs (Sp,Sp)-(2-(P(S)Ph2)FcCH2)2S resultierte. In Anwesenheit elektronenreicher Aromaten wurden elektrophile aromatischen Substitutuionen und die Bildung chiraler ferrocenylmethyl-substitutuierter Benzene beobachtet. Deren Einsatz als Ligand ergab Biaryle mit bis zu 26 % ee.