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The concept of bioreductive prodrug chemotherapy is introduced in chapter 1. Tumour cell hypoxia is a significant factor in limiting tumour growth control with conventional radiotherapy and some chemotherapeutic agents. Following therapy these cells can repopulate and cause a relapse of the cancer. On the other hand, hypoxia is unique to tumours, and is therefore potentially exploitable. Bioreductive prodrugs are compounds in which a oxygen inhibited redox-based bioactivation step triggers a reaction leading to a lethal intermediate. The concept of bioreductive DNA alkylators and DNA topoisomerase 11 inhibitors is discussed. The synthesis of model thiazolylindole compounds based on the natural product BE \0988 are discussed in chapter 2. Two strategies were employed for the construction of the thiazolylindoles: the Hantzsch reaction; and nucleophilic substitution on 2-bromothiazole by an indolyl anion. The synthesis of thiazolylindolequinone compounds are discussed in chapter 3. The quinone C(5) position of the thiazolylindolequinone analogues was elaborated to provide a series of cyclic and acyclic C(5)-amino derivatives. Synthetic strategies towards the synthesis of indole-2-carboxylates are discussed in chapter 4. The Moody-Rees and Cadogan-Sundberg reactions were employed to provide a synthesis of the useful highly substituted indole [154]. The Brederek imidazole reaction (i.e., the reaction of a amidine and a-halo ketone) is discussed in chapter 5. Application of the Brederek reaction was employed towards the construction of the bisindole imidazole natural compounds, the nortopsentins. The biological properties of the compounds of the compounds synthesised are discussed in chapter 6. The compounds were tested for DNA topoisomerase 11 inhibitory activity and cytotoxicity under a hypoxic environment.

This study evaluated comparative genomic hybridisation (CGH) as a tool to detect candidate regions of the genome associated with chemoresistance. Using a variation on conventional CGH, DNA from three cell lines that were resistant to thymidylate synthase (TS) inhibitors (tomudex [TDX] or 5-fluorouracil [5-FU]) and their sensitive parent cells were evaluated. In MCF-7 and H630, cells that were resistant to TDX, a specific TS inhibitor with no other known cytotoxic potential, only a single region of change (18p gain) was apparent. The third cell line H630R10, which was resistant to 5-FU, had changes in several genomic regions following the acquisition of resistance, including 18p. Gain in the chromosomal region containing the TS gene (18pll.32) was detected by CGH in all three resistant cell lines. However, additional novel regions of interest were identified in the cells that were resistant to 5-FU, a cytotoxic agent known to have several other modes of cytotoxicity besides TS inhibition. These results suggested that CGH is of potential use in the detection of regions of the genome involved in chemoresistance. Having shown the potential of CGH as a tool for assessing chemoresistance at the genomic level, steps toward clinical application of this technique were evaluated. A prerequisite for study in archival pathology samples was successful DNA extraction and universal amplification of tumour DNA from paraffin-embedded tumour sections for CGH analysis. Degenerate oligonucleotide primed - polymerase chain reaction (DOP-PCR) was performed on minute quantities (50ngs) of fresh cell line DNA (H630R10) and tumour DNA (osteosarcoma), as well as paraffin-embedded DNA from the same case. The results of these DOP-PCR CGH reactions were compared with conventional CGH using l|0.g quantities of fresh DNA from both H630R10 cell line and osteosarcoma. The CGH profiles of the conventional CGH and DOP-PCR CGH did not show a high level of concordance, only 55% of the gains and 83.3% of losses detected by conventional CGH were detected by DOP-PCR CGH The use of universal amplification by DOP-PCR in paraffin-embedded sections was not taken forward into clinical evaluation. A study of colorectal cancer (CRC) was initiated which involved the microdissection of 29 Dukes' C CRC tumours from fresh frozen material for CGH analysis. This conventional CGH analysis of CRC tumours involved assessing each tumour twice by reversal of fluorochromes. Only genomic regions that were detected as changed in both forward and reverse profiles were accepted. This approach detected several regions of genome as changed across the 29 tumours. In all, 108 gains (a mean number of 3.7 aberrations per tumour, range 1-12) and 85 losses (a mean number of 2.9 aberrations per tumour, range 0-11) were detected in the 29 tumours. CGH analyses identified certain chromosomal regions as more likely to be changed than others. The most frequent aberrations detected across the 29 tumours was a loss of chromosomal arm 18q, seen in 31% of the tumours assessed. Gain was also common at some sites throughout the genome, for example, gain of chromosomal arms, 13q and 20q was seen in 27.6% of cases. Mann-Whitney U tests investigating the association between specific chromosomal aberrations such as gain of 20q or loss of 18q and known markers of CRC tumourigenesis (p53, p27, p21, Rb, cyclin Dl, PCNA, P-catenin, e-cadherin, c-erbB-2, bcl2, EGFR and c-erbB-2) assessed by immunohistochemistry (IHC) in 29 tumours found no association. Testing of the total number of genomic aberrations detected (loss + gain = genetic grade) rather than the frequency of aberration at specific chromosomal loci also found no association with the CRC tumour markers. Finally, the association between the chromosomal aberrations detected by CGH was investigated in relation to patient survival. This thesis has demonstrated the value of a global approach to the study of chemoresistance and tumourigenesis through the application of powerful technology such as CGH.

The phenomenon of multidrug resistance (MDR) in cancer cells is generally associated with P-glycoprotein (P-gp) expression and presents an obstacle to successful chemotherapy. Attempts to overcome P-gp-associated MDR using P-gp modulators, such as verapamil, have been hindered by their intrinsic in vivo toxicity. In 1991, however, Scala et al. demonstrated the alteration of P-gp function by interferon-alpha (IFN-α) in vitro at non-toxic in vivo concentrations, suggesting a basis for the use of IFN-α clinically in patients exhibiting P-gp-associated MDR. Drug resistance in B-CLL has been linked to the phenomenon of MDR, however, publications regarding this have been conflicting. The contrasting results prompted further investigation of the role of P-gp-associated anthracycline resistance and, using isolated β-lymphocytes from B-CLL patients, this investigation examined P-gp expression, function and IFN-α modulation in vitro. Optimum conditions for in vitro analysis of P-gp-associated anthracycline resistance were determined by examining the stability of the anthracycline, daunorubicin, in varying cell culture conditions. The resulting system balanced conditions affecting drug stability with those affecting cell survival. While other investigations have neglected the issue of drug stability, this study demonstrates that the instability of daunorubicin may be a critical variable determining the outcome of drug sensitivity studies. In RPMI + 2mM L-glutamine and 10% (v/v) FBS, loss of drug concentration is due to both adsorption and degradation and these experiments show that the presumed availability of drug may be over-estimated in in vitro studies. Furthermore, the degradation products might interfere with P-gp function and modulation. MDRl gene mRNA was detected in the B-cells of forty-three out of fifty B-CLL patients analysed, whereas P-gp expression, as measured by flow cytometry, resulted in only sixteen patients out of fifty-five being classed as positive (> 10% increase in staining as compared to the control). P-gp functionality and modulation studies on the B-cells of eleven patients confirmed the existence of an efflux mechanism with identical characteristics to P-gp using verapamil, the dye rhodamine 123 (rho123) and daunorubicin. Four patients were classed as functional low expressers (functional P-gp with low P-gp expression (7-10% increase in staining)), six were classed as functional high expressors (functional P-gp with high P-gp expression (20-57% increase in staining)) and one as a non-functional high expressor (non-functional P-gp with high P-gp expression (13.4% increase in staining)). Verapamil modulated rho123 efflux in all ten patients classed as P-gp functional expressors, and daunorubicin efflux in eight of these patients. However, IFN-α modulated rho123 and daunorubicin efflux in only two and one patients, respectively, even at concentrations higher than 500I.U./ml. In contrast to Scala et al. (1991), this finding suggests that at a well tolerated concentration IFN-α may not be suitable for use as a P-gp modulating agent in vivo in B-CLL, although conclusive evidence would require a larger study.

The present thesis, entitled Chemotherapy-induced cognitive changes, is being submitted in the alternative format, by Oana Calina Lindner to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Medical and Human Sciences, School of Psychological Sciences. The thesis consists of five empirical studies, written in article formats and three connecting chapters. The General introduction in Chapter 1, places the thesis in the context of late effects research in cancer survivors. I describe the prevalence of physical and emotional late effects, before going into more details on cognitive late effects. Chapter 2 provides a meta-analytical summary of cognitive impairments following chemotherapy in adult patients. It has already been published in Neuropsychology in 2014. Chapter 3 describes the general objectives and hypotheses of the empirical studies, and Chapter 4 provides more details on the General methods utilized in all the studies. The studies focus on pre- and post-treatment young adult cancer patients who were compared to age-, sex-, and education-matched controls. The instruments include a comprehensive neuropsychological battery, a newly designed memory task, and a complex battery of self-assessment questionnaires. Chapter 5 is the first empirical study, which will be submitted to Journal of Clinical Oncology. It describes the pattern of neuropsychological status of young adult cancer patients following treatment for lymphoma, sarcoma, breast cancer, and germ cell tumour. The impairments were specific to executive functioning, verbal memory, and visuospatial abilities. Uniquely, the chapter depicts differences between cancer groups. Because chemotherapy may not be the primary factor triggering such effects, Chapter 6 details the neuropsychological profile of a group of young adult pre-treatment patients diagnosed with the same malignancies. This chapter will be submitted to Journal of Neuropsychology. Impairments were observed on tests of attention, executive functioning and visuospatial abilities. Both Chapters 5 and 6 emphasize the importance of matching on full scale IQ in cross-sectional studies and they provide evidence that patients' performance on tests of verbal memory and executive functioning may vary as a function of age. Chapter 7 will be submitted to Psycho-Oncology and it suggests the presence of acute memory deficits after the first treatment. Finally, Chapter 8, which will be submitted to Psychosomatic Medicine, provides an in-depth description of the psycho-emotional status of cancer survivors. It describes higher levels of anxiety, depression, fatigue, and cognitive complaints, which mediated the relationship between illness perceptions and quality of life. The complex interaction between these psycho-emotional factors is interpreted within the framework of cognitive-behavioural therapies, which may provide a method to decrease the emotional burden of survivorship in clinical practice. Finally, Chapter 9 summarizes all the empirical findings whilst connecting them to previous literature and specifying future research direction.

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