Funkční analýza hmyzích reduktáz a desaturáz mastných acylkoenzymů A

Lipidy odvozené od mastných kyselin představují důležité biomolekuly. Kromě své hlavní role při stavbě buněk, ukládání energie a signalizaci, slouží celé řadě dalších funkcí, např. jako izolační či nesmáčivé povrchy, obranné látky a feromony. Na úpravách intermediátů vznikajících při biosyn- téze mastných kyselin se podílí celá řada enzymů, mezi něž se řadí např. i desaturázy, které syn- tetizují nenasycené mastné kyseliny, a reduktázy, jež syntetizují mastné alkoholy. Funkčně vysoce diverzifikované desaturázy a reduktázy z hmyzu, jakožto jedněch z nejhojněji zastoupených zvířat na světe, představují podstatný zdroj pro moderní biotechnologii buněčných továren. Tato práce shrnuje dostupné informace o reduktázách a desaturázách a popisuje výsledky, které jsme při jejich studiu získali. U čmeláků jsme identifikovali několik reduktázových transkriptů, jež jsou hojně zastoupeny v samčí feromonové žláze. Příslušné enzymy jsme následně funkčně charakterizovali v kvasinkách a odhadli jejich zapojení do biosyntézy mastných alkoholů v tkáni produkující značkovací feromon. Studované enzymy redukují širokou řadu substrátů, od krátkých mastných acylů (C14) až po velmi dlouhé (C26), od nasycených mastných acylů po polynenasycené. Dále jsme poukázali na to, že na expanzi a následném funkčním rozrůznění čmeláčích...Fatty acid-derived lipids are an important class of biomolecules. In addition to their primary role in cellular architecture, energy storage and signaling, they function in many other ways, e.g. as isolating or waterproof coatings, defense compounds and pheromones. Multiple enzymes mod- ify intermediates originating from fatty acid biosynthetic pathway, including desaturases (FADs) which synthesize unsaturated fatty acids, and reductases (FARs) which synthesize fatty alcohols. Functionally highly diversified fatty acyl-modifying enzymes from insects, one of the most abun- dant animals in the world, present a significant source for modern biotechnology of cell factories. This thesis summarizes available information on the FADs and FARs and describes the results which we have obtained while studying them. In bumblebees, we identified several FAR transcripts which are abundantly expressed in male pheromone-producing tissue. We then functionally characterized the corresponding enzymes in yeasts, and estimated their participation in the biosynthesis of fatty alcohols observed in the mark- ing pheromone-producing tissue. The studied enzymes reduce broad range of substrates, from short fatty acyls (e.g. C14) to very long ones (e.g. C26), from saturated fatty acyls to polyunsatu- rated ones. We also found...Katedra biochemieDepartment of BiochemistryPřírodovědecká fakultaFaculty of Scienc

Production of Long Chain Fatty Alcohols Found in Bumblebee Pheromones by Yarrowia lipolytica

Fatty alcohols (FA-OH) are aliphatic unbranched primary alcohols with a chain of four or more carbon atoms. Besides potential industrial applications, fatty alcohols have important biological functions as well. In nature, fatty alcohols are produced as a part of a mixture of pheromones in several insect species, such as moths, termites, bees, wasps, etc. In addition, FA-OHs have a potential for agricultural applications, for example, they may be used as a suitable substitute for commercial insecticides. The insecticides have several drawbacks associated with their preparation, and they exert a negative impact on the environment. Currently, pheromone components are prepared mainly through the catalytic hydrogenation of plant oils and petrochemicals, which is an unsustainable, ecologically unfriendly, and highly expensive process. The biotechnological production of the pheromone components using engineered microbial strains and through the expression of the enzymes participating in the biosynthesis of these components is a promising approach that ensures ecological sustenance as well. The present study was aimed at evaluating the production of FA-OHs in the oleaginous yeast, Yarrowia lipolytica, with different lengths of fatty-acyl chains by expressing the fatty acyl-CoA reductase (FAR) BlapFAR4 from B. lapidarius, producing C16:0-OH, C16:1Δ9-OH, and lower quantities of both C14:0-OH and C18:1Δ9-OH, and BlucFAR1 from B. lucorum, producing FA-OHs with a chain length of 18–26 carbon atoms, in this yeast. Among the different novel Y. lipolytica strains used in the present study, the best results were obtained with JMY7086, which carried several lipid metabolism modifications and expressed the BlucFAR1 gene under the control of a strong constitutive promoter 8UAS-pTEF. JMY7086 produced only saturated fatty alcohols with chain lengths from 18 to 24 carbon atoms. The highest titer and accumulation achieved were 166.6 mg/L and 15.6 mg/g DCW of fatty alcohols, respectively. Unlike JMY7086, the BlapFAR4-expressing strain JMY7090 produced only 16 carbon atom-long FA-OHs with a titer of 14.6 mg/L

Desaturase specificity is controlled by the physicochemical properties of a single amino acid residue in the substrate binding tunnel

Membrane fatty acyl desaturases (mFAD) are ubiquitous enzymes in eukaryotes. They introduce double bonds into fatty acids (FAs), producing structurally diverse unsaturated FAs which serve as membrane lipid components or precursors of signaling molecules. The mechanisms controlling enzymatic specificity and selectivity of desaturation are, however, poorly understood. We found that the physicochemical properties, particularly side chain volume, of a single amino acid (aa) residue in insect mFADs (Lepidoptera: Bombyx mori and Manduca sexta) control the desaturation products. Molecular dynamics simulations of systems comprising wild-type or mutant mFADs with fatty acyl-CoA substrates revealed that the single aa substitution likely directs the outcome of the desaturation reaction by modulating the distance between substrate fatty acyl carbon atoms and active center metal ions. These findings, as well as our methodology combining mFAD mutational screening with molecular dynamics simulations, will facilitate prediction of desaturation products and facilitate engineering of mFADs for biotechnological applications

Software pro rozvrhování operací a evidence nástrojů pro nemocniční zařízení

Nowadays in most medical facilities is daily schedule of operations mainly created manually or with minimal support of software tools. Result of this is occasional ineffectivity of operation schedule and primarily huge time consumption for surgery chief who could use his time for more important things related to his work. Another reason for creating this application is that information about patients, operations, wards and diagnosis are stored decentralized in different applications and systems, which makes working with them more difficult. Target of this application is implementation application for creating operating schedules in hospital facilities with assurance of necessary instruments, wards and instrument evidence. Where all data about all necessary entities will be stored and managed centralized. Purpose of final program is make schedule creating more effective and partially automatized operation planning

Functional analysis of insect fatty acyl-coenzyme A reductases and desaturases

Fatty acid-derived lipids are an important class of biomolecules. In addition to their primary role in cellular architecture, energy storage and signaling, they function in many other ways, e.g. as isolating or waterproof coatings, defense compounds and pheromones. Multiple enzymes mod- ify intermediates originating from fatty acid biosynthetic pathway, including desaturases (FADs) which synthesize unsaturated fatty acids, and reductases (FARs) which synthesize fatty alcohols. Functionally highly diversified fatty acyl-modifying enzymes from insects, one of the most abun- dant animals in the world, present a significant source for modern biotechnology of cell factories. This thesis summarizes available information on the FADs and FARs and describes the results which we have obtained while studying them. In bumblebees, we identified several FAR transcripts which are abundantly expressed in male pheromone-producing tissue. We then functionally characterized the corresponding enzymes in yeasts, and estimated their participation in the biosynthesis of fatty alcohols observed in the mark- ing pheromone-producing tissue. The studied enzymes reduce broad range of substrates, from short fatty acyls (e.g. C14) to very long ones (e.g. C26), from saturated fatty acyls to polyunsatu- rated ones. We also found..

Komponenty pro vývoj firemních aplikací na platformě Android

V dnešní době rozmachu mobilních technologií stále více firem vybavuje svoje pracovníky v terénu (obchodní zástupce, pojišťovací agenty, servisní pracovníky apod.) mobilními aplikacemi pro zefektivnění jejich práce. Na trhu existuje několik univerzálních aplikací, které však vyžadují použití nějakého middleware anebo je nelze dostatečně modifikovat pro specifické potřeby zákazníka. Zde přichází na řadu vývoj vlastní mobilní aplikace. Cílem této práce je zanalyzovat požadavky na takovéto aplikace a na základě toho navrhnout sadu znovupoužitelných komponent, které ulehčí vývoj takovýchto nativních aplikací na platformě Android. Součástí práce bude pilotní aplikace demonstrující použití jednotlivých komponent na vzorových datech. Powered by TCPDF (www.tcpdf.org)Today, in the era of mobile technology boom, more and more companies equip their employees working in the field (salesmen, insurance agents, service workers etc.) with mobile applications in order to improve the efficiency of their work. On the market, there exist several universal applications which, however, require using some middleware, or they cannot be sufficiently modified to the customer specific needs. In this case comes the development of custom mobile application. The aim of this work is to analyze the requirements for such applications and based on this analysis propose a set of reusable components that will facilitate the development of these native applications on the Android platform. This work will also include a pilot application demonstrating the use of individual components on the sample data. Powered by TCPDF (www.tcpdf.org)Department of Software EngineeringKatedra softwarového inženýrstvíMatematicko-fyzikální fakultaFaculty of Mathematics and Physic

Strategies for generalized Reversi

The aim of this work is to design generalized Reversi (also known as Othello) and implement the application for editing and testing strategies of artificial intelligence. In the beginning of the work full rules of the game and solved problems are presented. After that follows detailed description of implemented strategies, programmer and user manual. At the conclusion of the work there is the report about using the application and possible extensions of the application

Software pro rozvrhování operací a evidence nástrojů pro nemocniční zařízení

Nowadays in most medical facilities is daily schedule of operations mainly created manually or with minimal support of software tools. Result of this is occasional ineffectivity of operation schedule and primarily huge time consumption for surgery chief who could use his time for more important things related to his work. Another reason for creating this application is that information about patients, operations, wards and diagnosis are stored decentralized in different applications and systems, which makes working with them more difficult. Target of this application is implementation application for creating operating schedules in hospital facilities with assurance of necessary instruments, wards and instrument evidence. Where all data about all necessary entities will be stored and managed centralized. Purpose of final program is make schedule creating more effective and partially automatized operation planning

Chemo-enzymatic synthesis of antiviral prodrugs

Lipases have been widely applied in the manufacture of food products and in some areas of the industry, nowadays they are used in synthetic organic chemistry catalyzing the hydrolytic/esterification reactions under very mild conditions in the field of protecting groups or enantiomer resolution. In this study, the commercial lipase from bacterium Pseudomonas fluorescens was immobilized using the sol-gel process into organosilicate materials with propyl, octyl or phenyl substituents. The highest hydrolytic activity was found in the enzyme on the octyl-derived carrier. The immobilized enzymes differ in their hydrolytic activities on 4-nitrophenyl esters of various lengths. Subsequent experiments revealed quite good pH stability of the enzymes in a buffer (incubations in pH 3 through pH 11), as well as good temperature stability in isooctane (incubations at up to 100 řC). The majority of organic solvents seem to have no substantial effect on the lipase activity. The biocatalytic properties were studied on a model compound from the group of the acyclic nucleoside analogues - 9-(2',3'-dihydroxypropyl)adenine (DHPA). It was found for example that the best acyl donors are vinyl esters, that the lipase shows a preference towards longer vinyl esters, that the reaction proceeds faster in non-polar solvents or that it..