Recent studies have highlighted the poor specificity of a battery of in vitro genotoxicity tests when predicting rodent carcinogenicity, in some cases leading to misleading predictions of genotoxic carcinogenicity. GreenScreen HC (GSHC) is a highly sensitive human cell-based genotoxicity assay that, in contrast to the battery in vitro mammalian cell tests, also exhibits high specificity. GSHC employs the response of the human DNA damage-inducible gene GADD45a as a marker of genotoxic stress, using a GFP reporter construct hosted by the TK6 cell line. In order to better understand the biological basis for the high specificity of GSHC, three approaches were taken. Firstly, the relevance of the choice of a p53-competent host cell line, TK6, was investigated. A database was compiled consisting of published genotoxicity data from the in vitro battery tests performed in TK6 cells, and comparative GSHC data generated. This work revealed that discordance existed between the tests analysed, therefore the specificity of GSHC was not dictated by the choice of cell line alone. To confirm that misleading positive results could still be generated in the GSHC TK6 cell line, mutation assays were performed for compounds with pre-existing misleading positive battery test data, and a dose-dependent increase in mutation frequency was observed for 1 of 3 compounds. Secondly, the importance of wild-type p53 to the specificity of GSHC was investigated by generating p53-deficient GADD45a-GFP reporter cell lines. This work identified wild-type p53 as a key regulator of GADD45a in the context of GSHC, with reduced expression levels observed in response to genotoxic stimuli. Finally NF-kB, a putative direct regulator of GADD45a in response to DNA damage, was shown to contribute to the expression of GADD45a at high doses of 4-nitroquinoline-N-oxide via mutational analysis of two NF-kB binding sites in the promoter and intron 3 of the GADD45a-GFP reporter. This implicates NF-kB in the direct regulation of GADD45a in response to genotoxic stress in the context of GSHC. The work presented here demonstrates that the high specificity of GSHC is not dictated solely by the use of the p53-competent TK6 cell line, but is due to the use of a unique biosensor of genotoxic stress; GADD45a expression. Interference with the function of two key stress response signalling nodes, p53 and NF-kB, revealed that these regulators dictate the magnitude of expression of GADD45a, directly impacting upon the specificity of GSHC.

Phenolic antioxidants reduce the effect of oxidative stress within cells. They are found in a various fruits, vegetables and as food additives to reduce spoilage. Consumption of antioxidants by humans has been linked with increased lifespan and reduced incidence of cancer and cardiovascular disorders (Cabrera et al. 2006; Kuriyama 2008). In cultured mammalian cells however, some of these phenolic antioxidants have been reported to generate reactive oxygen species (ROS), leading to chromosomal breakage (Long et al. 2007; Long & Halliwell 2001). It is clear then, that amongst this group of compounds, in vitro toxicological study is not a reliable prediction of human hazard. It is for this reason that the work described in this thesis was undertaken: the principal aim was to gain a better understanding of the reasons underlying this contradiction. It has been suggested that excessive ROS generated in vitro might be a result of the higher levels of oxygen (~20%) compared to (1-7%) in vivo: (Yusa et al. 1984; Turrens et al. 1982). With clearer understanding, new experimental approaches might be taken to highlight or reduce positive in vitro genotoxicity test results that might be considered misleading. A diverse set of test compounds was first chosen. It included polyphenolic (PPA), monophenolic (MPA) and non-phenolic antioxidants (NPA), in addition to mechanistically characterised oxidants, genotoxins and cytotoxic, non-genotoxins as controls. Genotoxicity was assessed in vitro using the GADD45a, GFP reporter assay and in silico using Derek Nexus™. Amongst the 19 antioxidants assessed, the 11 of 12 of PPAs, 0 of 4 MPAs and 1 of 3 NPAs (ethoxyquin) produced positive results in vitro and 8 of 12 PPAs generated alerts of at least plausible genotoxicity in silico. To discover whether these results were the result of cellular hyperoxia-promoted generation of physiologically irrelevant ROS in cells, genotoxicity was reassessed in the presence of 1 and 5% oxygen. This reduced oxygen exposure had no effect upon the qualitative result for any of the assessed compounds and a negligible effect upon the dose at which any positive result was produced. An assessment of the ability of antioxidants to generate potentially genotoxic ROS within cells was carried out using the intracellular fluorescent dye, dichlorofluorescin diacetate (DCFH-DA). 10 of 12 PPAs, 0 of 4 MPAs and 1 of 3 NPAs (ethoxyquin) were shown to increase the level of ROS within TK6 human lymphoblastoid cells within 4 hours of compound exposure. Within this same timeframe, the mitochondrial membranes in cells treated with 10 of 12 PPAs, 2 of 4 MPAs and 1 of 3 NPAs (ethoxyquin) were shown to become depolarised using JC-1 dye. It was unclear however, whether mitochondrial membrane depolarisation was a cause or a consequence of ROS generation within the cells. In order to assess whether the increase in intracellular ROS led to an increase in oxidised DNA within treated cells, 8-oxoguanine (8-OG) was quantified using a FITC conjugated anti8-OG antibody. This assessment revealed that levels of the oxidised base were only increased in cells exposed to two of the 12 PPAs (quercetin and resorcinol). The level of 8OG detected was lower than the vehicle control for cells treated with 10 of the 15 antioxidants. One interpretation of this is that these agents induce the repair pathway for oxidative damage, which leads to a lower level of oxidised DNA bases in the genome. The results showed that while a large proportion of PPAs produce genotoxic results in vitro and lead to increased levels of ROS, the amount of oxidised DNA is not higher in treated cells. This would suggest the presence of a different mechanism for the observed genotoxicity.

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Definované markery tvoří základ určování polohy kamery pro velké množství aplikací s rozšířenou realitou, v případě že jsou přísné požadavky na rychlost a robustnost. Tato práce popisuje účinnou metodu pro určení pózy kamery pomocí Uniformního pole markerů a několik realistických aplikací na bázi popsané metody. Metoda je velice výpočetně levná a poskytuje spolehlivou detekci pro několik výpočetních platforem, včetně běžných chytrých telefonů. Markery jako část zobrazené informace na monitorech jsou použité v této práci pro určení relativní orientaci mezi poskytovatelem obsahu a užívatelským zařízením, sloužícím pro výběr prvků užívatelského rozhraní při  interakci a migraci úkolů. Ve filmařském průmyslu poskytuje popsaná metoda pro zjištění polohy kamery jako součást klíčovaní pozadí filmářům živý náhled virtuální scény. Výsledky ukazují, že popsaná metoda pro detekci pole markerů má srovnatelnou úspěšnost a přesnost v porovnání s ostatními metodami na bázi markerů a je několikrát rýchlejší. Aplikace zahrnuté v této práci podle výsledků testů jsou životaschopné - rychlejší a levnější - alternativy k existujícím řešením.