Chapter 1 reviews DNA structure and DNA molecular recognition by synthetic agents, including supramolecular helicates and cisplatin. An overview of various types of helicates and cisplatin anticancer drugs and their mechanisms of action is discussed. Examples of non-platinum anticancer drugs are also presented. In Chapter 2, the synthesis of novel metal-based supramolecular helicates and attempts to combine them with the cisplatin anticancer agent are described. In some cases X-ray crystallography data are presented and discussed in detail. In addition, the synthesis of the fluorescent europium helicate is described. In Chapter 3, stability and DNA binding properties of the synthesised metallo-helicates are investigated using UV/Vis spectroscopy, CD and LD techniques. The ability of the complexes to unwind plasmid DNA and stabilise DNA three-way junction formation is explored by gel electrophoresis experiments. The results demonstrate that the geometry and size of the helicates are crucial for DNA three-way junction recognition. In Chapter 4, biological evaluation of antineoplastic activity of the metallo-helicates synthesised in this work, as well as of the compounds supplied by other members of the group, is studied using an MTT colorimetric assay. The results of the study reveal that some of the complexes exhibit a potent cytotoxic activity.

Flow cytometry (FCM) was utilized for monitoring the physiology of \(E.\) \(coli\) cells in orange juice (OJ) as well as a model orange juice (MOJ). Compared to FCM, plate counts highly underestimated the true number of viable cells in OJ. As a part of this study, the effects of the change in major components of OJ on viability of the cells in OJ and MOJ was investigated using FCM. Increase in ascorbic acid and amino acid concentrations of MOJ improved both the culturability and FCM viability of the cells. FCM was also employed for studying the effects of OJ clarification on viability of \(E.\) \(coli\) in OJ. Although, reduction in cloud content of OJ increased the number of healthy cells, however, the removal of cloud particles of larger than 0.7 μm appeared to increase the antimicrobial efficacy of particles of smaller than 0.7 μm. The effects of washing E. coli cells with available chlorine, H\(_2\)O\(_2\) and organic acids on their subsequent viability in OJ was also investigated. While increase in concentration of sanitizers resulted in a significant reduction in healthy populations, the total number of viable cells either remained constant or increased particularly in case of H\(_2\)O\(_2\)-washed cells.

Cancer Chemotherapy treatment involves the administration of drugs to patients, these drugs mainly work by interacting with the cell cycle or inhibiting DNA synthesis. Unfortunately, the toxicity of these chemotherapy drugs is severe and can have serious side-effects on different tissues and organs of the body. In chemotherapy treatment about 85% of cancer patients exhibit some degree of liver or kidney damage. Therefore, the aim of this study was to investigate the cytotoxicity of some of the most commonly used chemotherapy drugs; Methotrexate (MTX), Etoposide, Cisplatin and Doxorubicin (DOX) on liver and kidney cell lines (HepG2, Huh7.5, COS-7 and HK2). Therefore, our focus were on studying the molecular mechanism by which these drugs cause cell death in liver and kidney cells. This study also investigated the effects of some Ca2+ binding proteins (RGN, SERCA1, SERCA2b, SPCA1a, SPCA2) to test their ability to decrease the toxicity of these chemotherapeutic drugs in liver and kidney cells. The results showed that Etoposide, Cisplatin and DOX induce cell death in both kidney and liver cell lines via several different pathways such as apoptosis, necrosis, and autophagy. The results presented here also showed that several of the drugs used induced cell death by a novel new autophagic pathway in liver and kidney cells. Our data also suggested that regucalcin (RGN) and the endoplasmic reticulum Ca2+ pumps (SERCA1 and SERCA2b), but not the secretory pathway Ca2+ pumps (SPCA1a and SPCA2) were able to protect against different types of chemotherapy-induced toxicity in liver and kidney cells. These new observations will help to build up our awareness of the diverse effect of these drugs have on liver and kidney cells and may also help to develop protective interventions and strategies in the future to reduce hepatotoxicity and nephrotoxicity caused by these drugs.

Birt Hogg Dubé (BHD) syndrome is an inherited cancer susceptibility syndrome characterised by the development of fibrofolliculomas on the face and upper torso, and increased risk of lung cysts, spontaneous pneumothorax and renal cancer. The findings presented in this thesis advance knowledge into how the mutations in the FLCN gene cause the phenotypes associated with BHD syndrome, and provides novel insights into the functions of folliculin within the cell. The results presented provide further evidence of the association between BHD syndrome and increased risk of colorectal cancer in a subset of BHD syndrome families, and suggest that this association appears restricted to those patients with an exon 11 mononucleotide tract mutation. Evolutionary conservation analysis across the FLCN sequence suggests that pathogenic mutations could be expected throughout the gene, and identifies a region between codons 100-230 of increased evolutionary significance. The experiments undertaken demonstrate a practical strategy for determining the pathogenicity of non-truncating folliculin variants in vitro, and indicate that loss of protein stability is the main mechanism of pathogenicity for the previously reported non-truncating mutations within FLCN. Finally, this thesis reports the first identification of p0071 as a folliculin interacting protein. Folliculin deficiency exerts a functional impact on previously reported p0071 functions inducing RhoA signalling upregulation, mitotic defects and disruption of cell junctions. These results demonstrate the potential efficacy of using inhibitors downstream of RhoA as therapeutic targets in BHD tumours with dyregulated RhoA signaling, and provide novel directions for research into BHD syndrome.

Clusterin (CLU) is a multifunctional glycoprotein widely expressed as two isoforms. One isoform, sCLU is secreted, cytoplasmic and anti-apoptotic, the other, nCLU, is nuclear and pro-apoptotic. Seven genes, DKK3, TIMP1, CADM1, AKAP12, KLF4, RNASET2 and CLU were identified to be candidate tumour suppressor genes in cervical neoplasia and subsequent validation led to an evaluation of the regulation and expression of CLU at three sites of squamous cancer: the oral cavity, cervix and nasopharynx. Down-regulation of CLU was demonstrated in nasopharyngeal cancer (NPC) and oral cancer and loss of one CLU allele and methylation of the other in the NPC cell line C666-1. This defect has been repaired in this NPC cell line and showed that overexpression of the nuclear isoform of CLU resulted in reduced proliferation and decreased cell viability. Overexpression of both isoforms of CLU in C666-1 cells and their knockdown in HeLa cells regulates NF-kB activity, with a stabilisation of IkB\(\alpha\) following CLU overexpression in C666-1. Although sCLU is now considered a promising therapeutic target because of its anti-apoptotic function, with an antisense oligonucleotide currently undergoing clinical evaluation, results suggest that further consideration needs to be given to the possible tumour suppressor function of nCLU.

I investigated the role of genetic, environmental and intrauterine factors in child development using data from two large twin studies; the East Flanders Prospective Twin Survey (EFPTS) and the Twins and Multiple Births Association Heritability Study (TAMBAHS). An association between birth weight and child development has already been established. Potential associations between other factors of the intrauterine environment and child development were investigated in this thesis. Heritabilities of the umbilical cord, IQ, temperament and behaviour problems were estimated. Fetal characteristics, such as birth weight, placental weight and morphology, umbilical cord knots, length and insertions were investigated in relation to cognitive development in the EFPTS study. The impact of maternal pre-pregnancy weight on temperament and behaviour problems was examined in the TAMBAHS study. High heritability estimates were observed for certain dimensions of the umbilical cord, temperament and IQ; for behaviour problems, genetic, shared and non-shared environment were important. Low birth weight and cord knotting was associated with lower IQ; an association was observed between maternal overweight and children aggressive behaviour. The results are discussed in the context of the Developmental Origins of Health and Disease (DOHaD) hypothesis, highlighting the role of the intrauterine environment in child development.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of several human malignancies. Herpesviruses are known to modulate cellular pathways responsible for the recognition and repair of DNA lesions, collectively known as the DNA damage response (DDR). Here it is demonstrated that lytic reactivation of KSHV in B cells results in activation of the ATM and DNA-PK kinases that regulate the response to DNA double-strand breaks (DSBs). This DDR does not depend on amplification of viral DNA and results in phosphorylation of downstream proteins involved in DNA repair, cell cycle regulation and apoptosis. Specific inhibition of ATM activity attenuates KSHV replication while, in contrast, abrogation of DNA-PK activity enhances amplification of viral DNA. It is also shown here that cells containing lytic virus enter S phase which is required for efficient viral replication and robust activation of the DDR. In addition, immunofluorescence microscopy reveals that DNA damage sensing proteins such as MRE11 and Ku80 localise to sites of KSHV replication while other DSB repair proteins form foci in cellular DNA. Specific inhibition of MRE11 exonuclease activity in B cells restricts KSHV replication efficiency indicating that this DDR protein contributes positively to this phase of the viral lifecycle.

The ability to generate a tumour-reactive T cell compartment is possible through the genetic engineering of patient T cells with tumour-reactive TCRs or CARs. The clinical testing of such therapies is garnering increasingly encouraging results, particularly in haematological malignancies. However, identification of more potent and specific target antigens, and combating immunosuppression in the tumour microenvironment is necessary to transfer these clinical responses to solid tumours. We investigated whether recurrent cancer mutations encode immunogenic neoantigens presented by common HLA class I alleles. We isolated TCRs specific for putative neoepitopes derived from common mutations in calreticulin (mCALR), and FBXW7. These TCRs showed moderate affinity but fine peptide specificity for mutant peptides, with some TCRs capable of recognising mutant cell lines. However, mass spectral analysis of MHC-eluted peptides and MHC class I tetramer staining of patient T cells suggested putative mCALR neoepitopes were not naturally processed and presented. Finally, we investigated whether ectopic expression of arginine recycling enzymes argininosuccinate synthetase (ASS) and/or ornithine transcarbamylase (OTC) in CAR T cells endows resistance to immunosuppression driven by arginine depletion. Increased ASS/OTC expression and function was detected in CAR-engineered T cells, however enhanced cytotoxicity and proliferation of CAR T cells was not observed following arginine depletion.

Cancer cells reprogram their metabolism to supply biosynthetic and bioenergetic demands of rapid proliferation. Microenvironmental changes, such as hypoxia, further influence tumour metabolism, driving malignancy. Recent identification of cancer-associated mutations in succinate dehydrogenase (SDH), fumarate hydratase and isocitrate dehydrogenase (IDH) have shown that genetic alterations can directly alter tumour cell metabolism, and may be required for malignant transformation. Mutations in these metabolic enzymes promote tumorigenesis by hijacking the adaptive response to hypoxia. Understanding the metabolic vulnerabilities associated with these mutations may therefore elicit the design of more selective therapies. Employing a combination of analytical approaches to study metabolism, the research objectives were to characterise metabolic vulnerabilities associated with cells mutated in SDHB and IDH1. Results show that cells deficient in SDH activity maintain proliferation and viability by increasing dependency on pyruvate carboxylase for de novo aspartate synthesis. Mutations in IDH1 have a complex role in the metabolic adaptation to hypoxia, partially compromising this hypoxic response, yet also demonstrating aspects of pseudohypoxia, such as increased proline anabolism. This thesis reveals a metabolic vulnerability that could be therapeutically targeted to treat SDH-mutated tumours, and a novel redox-sensitive metabolic pathway, exhibited by both pseudohypoxic SDH and IDH1 mutated tumours, used to retain metabolic plasticity.

Primary malignant bone tumours are rare cancers that are characterised by different genetic and epigenetic alterations. A functional epigenomic approach was combined with the Illumina HumanHT-12.v4-BeadChip expression microarray in three Ewing Sarcoma (ES) cell lines to identify genome-wide functional methylation changes in these cells and ES primary samples. This study revealed eight frequently methylated genes in ES patients’ samples, where NPTX2 and PHF11 promoter methylation was associated with poor patient prognosis. The second methylation study involved genome-wide DNA methylation profiling of chordoma samples using the Infinium-HumanMethylation450-BeadChip microarray. This study identified a list of 8,819 loci which were differentially methylated between chordomas and controls and eight genes which were differentially methylated between recurrent and non-recurrent chordoma samples. RNA sequencing (RNA-seq) analysis of primitive small blue round cell tumour (SBRCT) samples was also carried out in order to identify gene fusions in this type of cancer. Three different somatic gene fusions in SBRCT samples were identified using RNA-Seq (CRTC1-SS18;BCR-UPB1 and KHDRBS2-CIC). Moreover, two other gene fusions were identified in unpaired SBCRT samples. Overall, this study used high-throughput technologies to identify novel genetically and epigenetically altered genes in different types of bone sarcoma which may, therefore, provide unique insight into bone sarcoma tumorigenesis.