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Conventional treatments for prostate cancer have significant limitations making it difficult to control the disease. Cyclopropabenzindoles (CBI) are more biologically potent, stable and synthetically accessible analogues of cyclopropapyrroloindole (CPI) anti-tumour antibiotics, such as duocarmycin-SA and CC1065. A polymeric prodrug carrying a CBI drug attached to the polymeric backbone through a PSA cleavable linker peptide has two modes of selectivity: activation by PSA and the EPR effect. To synthesise a 5-amino-seco-CBI analogue, 2,4-dinitronaphthalen- 1-ol gave di-Boc-1-iodonaphthalene-2,4-diamine in five steps (triflation, SNAr displacement with iodide, reduction (loss of iodine), protection and restoration of the iodine. For the amino-seco-CBI, it was important to discriminate between N2 and N4. Acidic removal of the Boc-group(s) resulted in deiodination. NMR investigations showed an unexpected Wheland-like cationic intermediate. N3 of naphthalene-1,3-diamine was selectively trifluoroacetylated and N1 was masked with Boc. Electrophilic iodination gave an orthogonally protected 1-iodonaphthalene-2,4-diamine. Allylation at the trifluoroacetamide was followed by free radical cyclisation with TEMPO trap. Removal of the trifluoroacetyl group allowed coupling to 5-(2-(dimethylamino)ethoxy)-1H-indole-2-carboxylic acid. Reductive removal of 2,2,6,6-tetramethylpiperidine, substitution of the exposed hydroxy group with chloride and removal of the Boc-group gave the amino-seco-CBI drug, 5-amino-1-chloromethyl-3-(5-(2-dimethylaminoethoxy)indole-2-carbonyl)-2,3-dihydro-1H-benz[e]indole. A DNA-melting assay confirmed that it binds very strongly to dsDNA causing a 13 deg. C increase in melting temperature. The drug was a highly potent cytotoxin in vitro, with IC50 = 18 nM against LNCaP prostate cancer cells. The polymeric prodrug system involved the synthesis of the pentapeptide SSKLQ. The amide side chain of glutamine can be masked as the nitrile and this can be quantitatively hydrated to the γ-carboxamide of L-Gln with hydroperoxide. The pentapeptide was coupled to 4-methoxynaphthalen-1-amine and to poly(ethylene glycol) as a model polymeric prodrug system. Efficient release of the model drug from the polymeric prodrug by PSA will allow this polymeric prodrug system to be adopted for the synthesised amino-seco-CBI drug.

Epidemiology of head and neck cancers is currently intensively studied topic. Recent shift in the age of incidence towards younger population is generally attributed to growing proportion of head and neck cancers caused by human papillomavirus - sexually transmitted virus which causes asymptomatic, but sometimes persisting infection that can lead to malignant transformation of the infected tissue. Significance of the topic lies mainly in the prognostic ad- vantage of patients with virally induced head and cancers and preventabil- ity of infections with human papillomavirus. First aim of this thesis is to demonstrate epidemiological trends of head and neck cancers in the Czech Republic, with the focus on change of age-specific incidence and mortality in the last three decades. Second aim of this thesis is to identify risk factors of oral infections with human papillomaviruses and head and neck cancer in a case-control and cross-sectional study of a hospital-based cohort. 1

Systemic administration of adenovirus type 5 (Ad5) vectors for the treatment of cancer is limited by poor circulation kinetics and inefficient uptake from the bloodstream into tumours. This study reports a novel method for linkage of highly-PEGylated gold nanoparticles (AuPEG) to Ad5 by a single reduction cleavable bond. The resulting ‘dandelion’ structure provides very effective steric shielding with only minimal and reversible modification of the Ad5 capsid. This ablates in vitro cell infection, improves protection against the binding of antibodies, and enhances in vivo circulation kinetics. Focused ultrasound is a promising technology for the non-invasive, targeted treatment of cancer. In the context of drug delivery, cavitational energy generated upon exposure of ultrasound contrast agents to focused ultrasound can be used as a powerful stimulus to move therapeutics over distances of hundreds of microns away from blood vessels. In addition to providing a platform for effective stealthing, conjugation of AuPEG to Ad5 also increases the effective density of Ad5. This increase in density imparts a second major advantage on the strategy, observed for the first time in the present study: denser particles are transported significantly farther by cavitation-induced microstreaming than identically-sized particles of lower density. Specifically, in in vitro tests using a tumour-mimicking flow-channel phantom model and in in vivo experiments using tumour bearing mice, Ad5–AuPEG was delivered farther from vessels in response to ultrasound induced cavitation than either naked Ad5 or polymer-coated Ad5. The enhancements in stealthing and improvements in response to ultrasound provided by this strategy enabled up to 12% (S.D. 0.97) of the injected dose to be deposited in the tumour, compared to just 0.12% (S.D. 0.05) for Ad5 without ultrasound (p < 0.001). Consequently, in a survival study, mice treated with Ad5–AuPEG with focussed ultrasound had the slowest tumour growth and longest survival rate when compared to mice treated with Ad5 alone, Ad5–AuPEG alone, or Ad5 with focussed ultrasound. These results provide compelling evidence that the combination of focussed ultrasound with density-augmented stealthed Ad5 results in improved delivery to tumours and therapeutic efficacy. This combination of ultrasound with particle modification for optimal cavitation-mediated delivery has the potential to be applied to a broad range of anti-cancer nano-medicines and therapeutics to augment their bio-availability for improved cancer treatment.

The canine transmissible venereal tumour (CTVT) is a contagious cancer that is naturally transmitted between dogs by the allogeneic transfer of living cancer cells during coitus. CTVT first arose several thousand years ago and has been reported in dog populations worldwide. The goals of this Thesis were (1) to gain further understanding of CTVT distribution patterns and prevalence around the world, (2) to use genetics to trace the historical spread of CTVT and (3) to map the genetic as well as phenotypic diversity of CTVT tumours around the world. To understand the distribution patterns of CTVT, I obtained information from 645 veterinarians and animal health workers in 109 countries, and generated a snapshot of the locations in which this disease is found. Additionally, as preparation for further genetic analysis, I collected samples from over one thousand CTVT cases from more than 50 countries, optimised methods for high-throughput DNA extraction and quantification and optimised a qPCR-based assay for CTVT diagnosis and host contamination detection. With the goal of tracing the historical spread of CTVT and learning about the genetic diversity of this disease, I sequenced complete mitochondrial genomes of 449 CTVT tumours and their matched hosts. The analysis of the CTVT mitochondrial diversity revealed that CTVT has captured mitochondrial DNA (mtDNA) through horizontal transfer events at least five times during the history of the lineage, delineating five tumour clades. CTVT appears to have spread rapidly around the world within the last 2,000 years, perhaps transported by dogs travelling along historic maritime trade routes. This work indicated that negative selection has operated to prevent accumulation of deleterious mutations in captured mtDNA, and that recombination has caused occasional mtDNA re-assortment. A histology-based screen of CTVT clades did not show any significant phenotypic differences between groups. In order to determine how the five mtDNA clades relate to each other, I analysed data from 539 CTVT exomes. This revealed that a single canine mtDNA haplogroup has recurrently and recently undergone multiple horizontal transfer events. Analysis of this haplotype highlighted a number of candidate genetic variants which may be conferring a selective advantage to this haplotype in CTVT, possibly by influencing mtDNA transcription or replication. Overall, genetic and phenotypic analysis of CTVT tumours from across the globe has broadened our understanding of CTVT diversity, and provided important insights into the biology of a unique transmissible cancer.