During the last decade, there is an increasing interest in applying deep learning in de novo drug design. In this thesis, a tool is developed to address the specific needs for generating small library for lead optimization. The optimization of small molecules is conducted given an input scaffold with defined attachment points. Various chemical fragments are proposed by the generative model and reinforcement learning is used to guide the generation to produce a library of molecules that satisfy user-defined properties. The generation is also constrained to follow user-defined reactions which makes synthesis controllable. Several experiments are executed to find the optimal hyperparameters, make comparison of different learning strategies, demonstrate the superiority of slicing molecules based on defined reactions compared to RECAP rules, showcase the model’s ability to follow different synthetic routes as well as its capability of decorating scaffolds with various attachment points. Results have shown that DAP learning strategy outperforms all other learning strategies. The use of reaction based slicing is superior than utilising RECAP rules slicing, it helps the model to learn the reaction filter faster. Also, the model was capable of satisfying different reaction filters and decorating scaffolds with various attachment points. In conclusion, the model is able to rapidly generate a molecular library which contains a large number of molecules sharing the same scaffold, with desirable properties and can be synthesised under specified reactions.

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Diploma thesis suggests a new functional use of former vocational boarding school near town center of Rousínov and deals with its reconstruction and modification to serve the new purpose. Current conditions of communication routes and public welfare are being analyzed in order to select proper urban model, that would be applied. Two buildings of former dormitories are to be replaced by leisure time activity center and multifunctional house with commercial space and retirement house respectively. The purpose of former canteen will be left as it this, although it is meant to be enhanced by adding cafeteria with roof garden. In the given context, an architectural design was elaborated while using former structures and layout but with new inner structure. Vertical wooden structures with dynamically spaced elements are used as a common feature. Apart from design of the buildings themselves a special care was also taken while creating the environment. Parks are laid out in a polygon grid, shaped by the straight lines of communication routes.

Cílem diplomové práce je návrh síťové konfigurační knihovny s důrazem kladeným na přenositelnost mezi operačními systémy na bázi Linuxu a BSD a rozšiřitelnosti podpory knihovny. V druhé kapitole práce zkoumá dostupné konfigurační rozhraní obou operačních systémů. Detailně pak rozebírá vlastnosti rozhraní Netlink socketů, které je primárním konfiguračním rozhraním pro síťové prvky na Linuxu, a systémové volání ioctl, které má na Linuxu menší schopnosti, ale zato je primárně používané na BSD a jiných UNIX systémech. Jsou též zkoumané rozhraní pro konfiguraci rozdílných firewallů. V třetí kapitole je práce zameřená na konkrétní typy síťových zařízení, specifika jejich konfigurace a jejich návaznost na rozhraní jádra popsané v druhé kapitole. V čtvrté kapitole jsou formulovány požadavky na konfigurační knihovnu: jednoduchá rozšiřitelnost, přenositelnost na různé operační systémy, podpora sledování změn a událostí a rozšiřitelnost o různé typy uživatelských rozhraní. Na základě výzkumu z předcházejících dvou kapitol je přednesen návrh knihovny. Návrh definuje konfigurační rozhraní jako hierarchii abstraktních tříd, oddělených od implementace. To umožnuje mít současně několik implementací stejného konfiguračního rozhraní i v rámci jednoho operačního systému. Jako vstupní rozhraní knihovny je definovaná třída LibNCFG, která má na starosti tyto konfigurační objekty vytvořit namísto uživatele. Tímto je dosažená jednoduchá rozšiřitelnost knihovny o nové rozhraní operačních systémů i o podporu konfigurace nových síťových prvků. Podpora pro nové uživatelské rozhraní se dá implementovat jako nová služba, která zabaluje rozhraní knihovny a poskytuje jiná rozhraní. Pro podporu sledování změn poskytuje třída LibNCFG metody pro registraci zpětných volání pro definované události. Ve čtvrté kapitole práce detailně popisuje rozhraní třídy LibNCFG, modulu Common a tříd NetDevice, EthDevice a BondDevice, které definují konfigurační rozhraní příslušných typů síťových zařízení. Pro tyto třídy jsou implementované konkrétní třídy NetlinkNetDevice, NetlinkEthDevice a sysfsBondDevice a popsané jejich implementační detaily. V páté kapitole je popsaná ukázková aplikace, která byla implementovaná pro účely předvedení jednoduchosti použití konfigurační knihovny. Nakonec jsou v závěru shrnuté výsledky práce a je vedena diskuze o možných vylepšeních a o pokračování projektu.

Fragment-screening by X-ray crystallography (XFS) is an expensive and low throughput fragment drug discovery screening method, and it requires a lot of optimization for each protein target. The advantages with this screening method are that it is very sensitive, it directly gives the three-dimensional structure of the protein-fragment complexes, and false positives are rarely obtained. The aim of this project was to help Sprint Bioscience assess if the advantages with XFS outweigh the disadvantages, and if this method should be used as a complement to their differential scanning fluorimetry (DSF) screening method. An XFS campaign was run using the oncoprotein vaccinia related kinase 1 (VRK1) as a target protein to evaluate this screening method. During the development of the XFS campaign, a diverse fragment library was created which consisted of 298 fragments that were all soluble in DMSO at 1 M concentration. The crystallization of the protein VRK1 was also optimized in this project to get a robust, high throughput crystallization set up which generated crystals that diffracted at higher resolution than 2.0 Å when they were not soaked with fragments. The soaking protocol was also optimized in order to reduce both the steps during the screening procedure and mechanical stress caused to the crystals during handling. Lastly, the created fragment library was used in screening VRK1 at 87.5 mM concentration with XFS. 23 fragment hits could be obtained from the X-ray crystallography screening campaign, and the mean resolution of the crystal structures of the protein-fragment complexes was 1.87Å. 11 of the 23 fragment hits were not identified as hits when they were screened against VRK1 using DSF. XFS was deemed as a suitable and efficient screening method to complement DSF since the hit rate was high and fragments hits could be obtained with this method that could not be obtained with DSF. However, in order to use this screening method a lot of time needs to be spent in optimizing the crystal system so it becomes suitable for fragment screening. Sprint Bioscience would therefore need to evaluate the cost/benefit ratio of using this screening method for each new project.

Syfte: Syftet med denna uppsats är att illustrera vilka effekter olika medietyper och medieteknologier har på biblioteksutformning samt på vilket sätt. Detta undersöks genom två grupper av frågeställningar där den ena fokuserar på medieteknologier som en aktör i ett nätverk och den andra hänvisar till medieteknologiers placering och effekten därav. Teori: För att besvara dessa frågeställningar används i uppsatsen tre huvudsakliga teorier som består av materiell media-teori, posthumanism samt aktör-nätverksteori (ANT). Metod: Den genomförda undersökningen är en fallstudie, och använder sig av en kombination av observationer, existerande dokument och semi-strukturerade intervjuer för att samla in ett empiriskt dataunderlag. Vidare används en kvalitativ innehållsanalys för att bearbeta det insamlade materialet. Slutsatser: Uppsatsen kommer fram till att processen att utforma ett fysiskt bibliotek kan karakteriseras som ett aktör-nätverk med ett stort antal aktörer som inverkar på bibliotekets design. Medier och medieteknologier är en av dessa aktörer som påverkar bibliotekets utformning genom deras kopplingar till andra aktörer, till värderingar och symbolism samt genom deras materiella förutsättningar.

La synthèse d’oligosides complexes reste difficilement réalisable par voie chimique. Le recours aux catalyseurs enzymatiques permettrait de pallier aux contraintes de la chimie mais les enzymes naturelles ne présentent pas toujours les propriétés adéquates et nécessitent d’être optimisées par ingénierie moléculaire. Le couplage de la chimie et de biocatalyseurs conçus « sur mesure », peut offrir une alternative prometteuse pour explorer de nouvelles voies de synthèse des sucres, notamment pour la mise au point de glycovaccins. L’objectif de cette thèse a ainsi visé à mettre en œuvre des stratégies d’ingénierie semi-rationnelles de l’amylosaccharase de Neisseria polysaccharea (ASNp), une α-transglucosylase utilisant le saccharose comme substrat, afin de concevoir de nouvelles spécificités de substrats et d’étendre le potentiel de cette enzyme à catalyser de nouvelles réactions, permettant ainsi d’aller bien au-delà de ce que la Nature peut offrir. Dans une première étude, une approche assistée par ordinateur a été suivie afin de remodeler le site actif de l’enzyme (sous-sites +1, +2 et +3) pour la reconnaissance et la glucosylation en α-1,4 d’un accepteur disaccharidique non-naturel (l’allyl 2-deoxy-2-N-trichloroacetyl-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranose). Le produit attendu, un trisaccharide, est un précurseur dans la synthèse chimio-enzymatique des oligosaccharides mimant les unités répétitives des lipopolysaccharides de Shigella flexneri, dont l’utilisation ultime est le développement de vaccins contre la Shigellose. Une approche computationnelle faisant appel à des outils dédiés au design automatisé de protéines et à une analyse des séquences a conduit au design d’une librairie d’environ 2.7x104 séquences, qui a ensuite été construite expérimentalement puis criblée. Au final, 55 variants actifs sur saccharose (le substrat donneur) ont été identifiés, et un mutant, appelé F3, a révélé sa capacité à glucosyler en α-1,4 le disaccharide cible. De manière étonnante, ce mutant possède 7 mutations au sein de son site actif, nécessaires au déploiement de sa nouvelle spécificité tout en maintenant son aptitude à utiliser le saccharose comme donneur d'unité glucosyle. Dans une deuxième étude, trois variants ont été identifiés lors du criblage de la librairie semi-rationnelle sur saccharose comme présentant de nouvelles spécificités de produits. Ces mutants ont été caractérisés plus en détails, ainsi que leurs produits, sur un plan biochimique et structural. Ces mutants, appelés 37G4, 39A8 et 47A10, contiennent entre 7 et 11 mutations dans leur site actif. Il a été montré qu’ils étaient capables de reconnaitre le saccharose et le maltose (un produit de la réaction avec le saccharose) comme donneur et accepteur pour synthétiser en quantités variables de l’erlose (α-D-Glucopyranosyl-(1→4)-α-D-Glucopyranosyl-(1→2)-β-D-Fructose) et du panose (α-D-Glucopyranosyl-(1→6)-α-D-Glucopyranosyl-(1→4)-α-D-glucose), des molécules non produites par l’enzyme sauvage. Des taux de production relativement élevés ont été obtenus pour ces molécules, dont les propriétés acariogènes et le pouvoir sucrant pourraient présenter un intérêt applicatif pour l’industrie alimentaire. Dans une dernière partie, un autre mutant, appelé 30H3, a été isolé lors du criblage primaire de la librairie de par son activité élevée sur saccharose (une amélioration d’un facteur 6.5 comparé à l’enzyme sauvage). Après caractérisation, le mutant s’est avéré synthétiser un profil unique de produits en comparaison de l’enzyme sauvage ASNp. Il s’est ainsi montré très efficace pour la synthèse de maltooligosaccharides solubles, de taille de chaînes contrôlée allant d’un DP 3 à 21, et de faible polydispersité. Aucun polymère insoluble n’a été identifié. La structure 3D du mutant résolue par cristallographie des rayons X a révélé un agrandissement de la poche catalytique en raison de la présence de 9 mutations introduites dans la première sphère.... / Chemical synthesis of complex oligosaccharides still remains critical. Enzymes have emerged as powerful tools to circumvent chemical boundaries of glycochemistry. However, natural enzymes do not necessarily display the required properties and need to be optimized by molecular engineering. Combined use of chemistry and tailored biocatalysts may thus be attractive for exploring novel synthetic routes, especially for glyco-based vaccines development. The objective of this thesis was thus to apply semi-rational engineering strategies to Neisseria polysaccharea amylosucrase (NpAS), a sucrose-utilizing α-transglucosylase, in order to conceive novel substrate specificities and extend the potential of this enzyme to catalyze novel reactions, going beyond what nature has to offer. In a first study, a computer aided-approach was followed to reshape the active site of the enzyme (subsites +1, +2 and +3) for the recognition and α-1,4 glucosylation of a non-natural disaccharide acceptor molecule (allyl 2-deoxy-2-N-trichloroacetyl-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranose). The trisaccharide product is a building block for the chemo-enzymatic synthesis of oligosaccharides mimicking the repetitive units of the Shigella flexneri lipopolysaccharides, and ultimately, for the production of a vaccine against Shigellosis disease. Using computational tools dedicated to the automated protein design, combined with sequence analysis, a library of about 2.7x104 sequences was designed and experimentally constructed and screened. Altogether, 55 mutants were identified to be active on sucrose (the donor substrate), and one, called mutant F3, was subsequently found able to catalyze the α-1,4 glucosylation of the target disaccharide. Impressively, this mutant contained seven mutations in the first shell of the active site leading to a drastic reshaping of the catalytic pocket without significantly perturbing the original specificity for sucrose donor substrate. In a second study, three variants were identified from the screening of the semi-rational library on sole sucrose as displaying totally novel product specificities. They were further characterized, as well as their products, at both biochemical and structural level. These mutants, called 37G4, 39A8 and 47A10, contained between 7 and 11 mutations into their active site. They were found able to use sucrose and maltose (a reaction product from sucrose) as both donor and acceptor substrates to produce in varying amounts erlose (α-D-Glucopyranosyl-(1→4)-α-D-Glucopyranosyl-(1→2)-β-D-Fructose) and panose (α-D-Glucopyranosyl-(1→6)-α-D-Glucopyranosyl-(1→4)-α-D-glucose) trisaccharides, which are not produced at all by parental wild-type enzyme. Relatively high yields were obtained for the production of these molecules, which are known to have acariogenic and sweetening properties and could be of interest for food applications. In a last part, another mutant 30H3 was isolated due to its high activity on sucrose (6.5-fold improvement compared to wild-type activity) from primary screening of the library. When characterized, the mutant revealed a singular product profile compared to that of wild-type NpAS. It appeared highly efficient for the synthesis of soluble maltooligosaccharides of controlled size chains, from DP 3 to 21, and with a low polydispersity. No formation of insoluble polymer was found. The X-ray structure of the mutant was determined and revealed the opening of the catalytic pocket due to the presence of 9 mutations in the first sphere. Molecular dynamics simulations suggested a role of mutations onto flexibility of domain B’ that might interfere with oligosaccharide binding and explain product specificity of the mutant.