3-Deoxy-D-arabino heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first step of the shikimate pathway, responsible for the biosynthesis of aromatic amino acids. This pathway is found in microorganisms, plants and apicomplexan parasites and its absence in mammals makes it a viable target for antimicrobial drug design. DAH7PS enzymes differ in the regulatory machinery that decorates the catalytic (β/α)8 barrel. Some DAH7PS enzymes are fused to chorismate mutase (CM), another enzyme in the shikimate pathway. This fusion protein is allosterically regulated by chorismate (CA) or prephenate (PA), the precursor of tyrosine and phenylalanine. It has been suggested that DAH7PS enzymes evolved these extensions to the core barrel for the sole purpose of regulation. Geobacillus sp DAH7PS (GspDAH7PSWT) is a thermophilic type Iβ DAH7PS enzyme with an N-terminal CM domain fused through a linker region. This thesis describes the functional characterisation work carried out on GspDAH7PSWT, in attempt to help determine how DAH7PS enzymes evolved such diverse methods of regulation. Chapter 2 describes the functional characterisation work carried out on the catalytic and regulatory domains of GspDAH7PSWT. The enzyme demonstrated both DAH7PS and CM activities with the DAH7PS domain determined to be metal dependent and most activated by Cd2+. PA completely inhibited the catalytic activity of GspDAH7PSWT, and AUC demonstrated an equilibrium exists between the dimeric and tetrameric quaternary states of the enzyme in solution. Chapter 3 describes the domain truncation of GspDAH7PSWT carried out at the linker region in order to obtain two separate protein domains, the catalytic domain lacking the N-terminal domain (GspDAH7PSDAH7PS) and the regulatory domain without the catalytic domain (GspDAH7PSCM). Both variants were fully characterised, and information obtained from each domain was compared to the respective catalytic and regulatory domains of the wild-type enzyme, which was also characterised. Like GspDAH7PSWT, GspDAH7PSDAH7PS showed greatest activation in the presence of Cd2+, with other metals having varying effects on activation rates and stability of the enzyme. Both truncated variants followed Michaelis-Menten kinetics where GspDAH7PSDAH7PS was found to be more active than GspDAH7PSWT and unaffected by PA, whereas GspDAH7PSCM was a less efficient catalyst than the CM domain of GspDAH7PSWT. AUC demonstrated that in solution an equilibrium occurs between the monomeric and tetrameric oligomeric states of GspDAH7PSDAH7PS. Chapter 4 summarises the findings of the thesis along with future directions of this research, combining the results obtained and expanding upon them. It is concluded that the catalytic regulatory CM domain supports both protein structure and allosteric regulation of GspDAH7PSWT

Předkládaná diplomová práce se zabývá extrakcí nanočástic oxidu titaničitého z produktů osobní péče, konkrétně opalovacích krémů a následnou charakterizací těchto částic. Počet komerčně dostupných produktů s obsahem nanočástic TiO2 se neustále zvyšuje a to se sebou přináší potřebu vyhodnotit potenciální osud a nepřímou expozici TiO2 nanošástic o různých velikostí a tvarů a zkoumat jejich celý životní cyklus. Bylo zkoumáno použití ultrafiltrace a ultracentrifugace jako extrakční metody. Dvě metody pro extrakci TiO2 nanočástic byly vyvinuty a aplikovány na vzorky opalovacích krémů. Extrahované částice mohou být použity pro ekotoxikologické studie, případně experimenty v mesokosmu. Velikost částic byla stanovena pomocí metody dynamického rozptylu světla a transmisní elektronové mikroskopie.