There is increasing evidence that the aberrant expression of cancer-testis (CT) antigens - a family of ca. 150 proteins that are both autoimmunogenic and mainly restricted to tumours in various types of human cancers - makes them attractive immunotherapy targets, as well as possible cancer diagnostic markers. We carried out a retrospective serological study of primary and secondary autoimmune responses of various cohorts of cancer patients prior to and/or following a variety of distinct treatments (chemotherapy, radiotherapy and immunotherapy), using a large number of archived human serum samples. Our goals were to develop and validate a novel cancer-testis and -associated antigen microarray platform and to then explore its utility and general applicability in the cancer immunology field. In addition, we sought to cross-correlate our protein microarray data from specific cohorts with in vitro T-cell re-stimulation assays for a selected subset of patients. Furthermore, as a means of determining the biological significance of our protein microarray data, we also collected clinical patient data where possible. The underlying hypothesis of our study was that there were measurable differences in autoantibody repertoires towards tumour-specific and -associated antigens between pre- and post-treated cancer patient samples (using various trial therapies), potentially augmented by prior chemo- or radiotherapy, which would correlate with likelihood of response of individual patients to a given therapeutic treatment - including those treatments that aim to generate T-cell responses - and which would also correlate with the nature and extent of individual patient responses to treatment.

Includes abstract. Includes bibliographical references (leaves 212-248).

Includes abstract. Includes bibliographical references (leaves 204-223).

Includes bibliographical references.

Includes abstract. / Includes bibliographical references (leaves 110-135). / Recent studies have revealed that interactions between tumour cells and the surrounding stroma play an important role in facilitating tumour growth and invasion. Stromal fibroblasts produce most of the extracellular matrix (ECM) components found in the stroma, including type I collagen. Previous in vivo studies in our laboratory have shown that type I collagen mRNA levels are decreased in stage II and III breast tumour tissue compared to adjacent normal tissue.

Angiotensin-I converting enzyme (ACE) is an important drug target in the treatment of heart disease due to its role in the regulation of blood pressure. ACE contains two domains, the N- and C-domains, both of which are catalytically active and heavily glycosylated. Glycosylation is one of the most important forms of post-translational modification, having a wide range of functions including protein folding, modulation of the immune response, and providing targeting signals. Glycosylation is required for the expression of active ACE and structural studies of ACE have been fraught with severe difficulties because of surface N-glycosylation of the protein. This problem has been addressed to a large extent with respect to the C-domain, where the role of glycosylation has been extensively characterised and a minimally glycosylated form was able to crystallise reproducibly. As yet, little is known about the degree and importance of N-linked glycosylation on the N-domain. The generation of minimally glycosylated N-domain, however, requires a greater understanding of the relative importance of the individual N-linked glycosylation sites.

The Fragile X-related disorders arise from an unusual mutation in the X-linked FMR1 gene. The mutation involves expansion, or an increase in the number of repeats, in a CGG•CCG repeat tract located in its 5' untranslated region. FMR1 alleles carrying 55-200 repeats are called Premutation (PM) alleles, and cause Fragile X associated tremor/ataxia syndrome (FXTAS) and Fragile X-associated primary ovarian insufficiency (FXPOI). FMR1 alleles having more than 200 repeats are referred to as full mutation (FM) alleles and cause Fragile X syndrome (FXS). These different alleles arise by intergenerational expansion of the repeat tract from smaller unstable alleles by a mechanism that is unknown. We have shown that in addition to germ line expansion, somatic expansion also occurs in a human cell line in vivo and in a FX PM mouse model. In the mouse model, we found that the extent of somatic instability is dependent on age, gender and tissue. Specifically, organs such as brain, liver and gonads are susceptible to expand more than heart and kidney and expansion is much more frequent in males than in females. No differences were found between male and female mice in the levels of the DNA repair proteins that had already been implicated in repeat expansion in model systems of other disorders thought to arise via a similar mechanism. Neither were there any differences between males and females in the amounts of proteins produced from X-linked DNA repair genes. We also showed that estrogen did not protect against expansion. However, we found that PM alleles expanded exclusively when they were located on the active X chromosome. Thus some of the differences between males and xii females in the level of somatic expansion might be due to the fact that females undergo X inactivation and thus have the PM allele on the inactive X chromosome in half (~50%) of their cells. It also indicates that transcription and/or an open chromatin configuration is required for expansion in the FX PM mouse.

Includes abstract. Includes bibliographical references (leaves 79-93).

Bibliography: leaves 133-156. / Cervical cancer is considered an important clinical problem in sub-Saharan Africa. Recent studies have suggested that epithelial tumors may be regulated by cyclooxygenase enzyme products. The purpose of this thesis was to determine the expression, localisation and possible functional role of cyclooxygenase enzymes in cervical carcinomas. The initial aim of the study was to determine whether cyclooxygenase-1 and cyclooxygenase-2 expession and prostglandin E₂ synthesis are up-regulated in cervical cancers. Real-time quantitative reverse-transcriptase polymerase chain reaction and Western blot analysis confirmed cyclooxygenase-1 and cyclooxygenase-2 ribonucleic acid and protein expression in all cases of squamous cell carcinoma and adenocarcinoma investigated. In contrast, minimal expression of cyclooxygenase-1 or cyclooxygenase-2 was detected in histologically normal cervix. Immunohistochemical analyses localised the site of cyclooxygenase-1 and cyclooxygenase-2 expression and prostaglandin E₂ synthesis to neoplastic epithelial cells of all squamous cell carcinomas and adenocarcinomas studied.

BisPMB (E, Z)-1,8-(Bis-p-methoxyphenyl)-2,3,7-trithiaocta-4-ene 7-oxide) is a synthetic analogue of the garlic compound ajoene. It is 12 times more active at inhibiting the growth of oesophageal squamous cell carcinoma WHCO1 cells and displays selectivity for cancer cells over normal cells. BisPMB is therefore attractive as a potential cancer therapeutic. In this study, bisPMB was found to inhibit WHCO1 cancer cell proliferation in a time and concentration dependent manner with 24 hour IC50's between 6.7 - 8.1 μM against a range of oesophageal cancer cell lines including WHCO1, KYSE30 and WHCO6. The normal oesophageal epithelial cell line, HET1A was found to be five times less responsive to bisPMB. Furthermore, bisPMB was found to induce apoptosis and G2/M cell cycle arrest in WHCO1 cells. Gene expression data obtained from the microarray analysis showed that bisPMB primarily targets the unfolded protein response (UPR) in WHCO1 cells. We also found that bisPMB deregulated the ER stress genes involved in protein processing in the endoplasmic reticulum and also deregulated MAPK pathways in WHCO1 cells. At a protein level, bisPMB was found to induce an increase in protein ubiquitination and in the expression of ER stress and UPR genes ATF4, Grp78 and CHOP in WHCO1 cells. We also observed a decrease in ATF6 90 kDa protein and transient XBP-1 mRNA splicing. The activation of p38, JNK and ERK MAPK pathways in bisPMB treated WHCO1 cells was also observed. Furthermore siRNA mediated knock-down of CHOP abolished the anti-proliferative effect of bisPMB in WHCO1 cells. However, inhibition of JNK and p38 MAPK by chemical inhibitors, SP600125 and SB 203580 respectively, had no effect on bisPMB antiproliferative activity against WHCO1 cells. On the other hand, inhibition of ERK1/2 MAPK by U0126 enhanced the anti-proliferative effect of bisPMB in WHCO1 cells. These results support the hypothesis that ER stress and MAPK signalling pathways are essential for bisPMB induced cytotoxicity in oesophageal cancer cells.