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.

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.

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.

CMV CD8+T-cell memory-inflation can occupy up to 50% of the total CD8+ T cell pool. Studies using an MHC class I immunevasion-deleted strain revealed novel peptide-epitopes across the virus genome both in-frame and translated in a non-canonical manner. This study functionally and phenotypically characterised CD8+T-cells responding to these CMV-derived epitopes with age. They were found to be frequent component of the in vivo repertoire dedicated towards HCMV during latency. A HLA-Cw*0702-restricted immunodominant CD8+T-cell response that accumulated within elderly donors was identified to reach 32% of the total CD8+T-cell pool producing IFN-γ/TNF-α. Subsequently, HLA-Cw*0702-restricted memory-inflation was observed to a further two peptides dominating the CD8+T-cell memory compartment. HLA-Cw*0702 CD8+T-cells demonstrated a TEMRA phenotype - CD45RA+/CD27-/CD28-/CCR7-/perforinhigh/granzymeBhigh - and represented promising candidates for inclusion in HSCT adoptive immunotherapies. Consequently, the global HCMV-specific CD8+T-cell response is being vastly underestimated by restricting studies to; in-frame translation products, HLA-A/-B-restricted peptide-epitopes and utilising WT-strains to characterise novel CD8+T-cell targets. Lastly, understanding why particular HCMV antigens induce inflationary CD8+T-cells will facilitate harnessing HCMV as a cancer therapy. In an attempt to direct HCMV-mediated inflationary responses towards malignancies, a HCMV-based vaccine vector expressing the NYESO1 CTAg was generated. Preliminary results indicate the immunogenicity of such a vaccine in vitro.

The 5-year survival rate of lung cancer patients is only 16%. As most patients are diagnosed at an advanced stage, little is known about early stages and mechanisms underlying the progression to metastatic disease. There are few targeted therapies available and targeting KRas driven lung cancer is especially challenging. KRAS is one of the most frequently mutated oncogenes in lung adenocarcinomas at ~33% of cases and is notably associated with resistance to EGFR inhibitors. In order to study tumour progression in vivo we chose a Cre/loxP inducible system in which Cre recombinase expressing Adenovirus is delivered to the lung by intranasal installation. In this model, Cre-mediated induction of a conditional KRasG12D allele gives rise to benign papillary adenomas (BPAs) that rarely progress to adenocarcinoma. Combined activation with conditional modest MYC overexpression however increases both the growth rate of the BPAs and their frequency of progression to adenocarcinoma. Deregulated MYC expression alone however gives rise to focal proliferation in the bronchioles but does not lead to tumours. Loss of functional Tp53 does not increase MYC’s tumour initiating potential in this model. Importantly, the KRasG12D/MYC model faithfully recapitulates the morphology of a subset of the human disease. I used Erk phophorylation status to distinguish between benign (p-Erk negative) and more advanced (p-Erk positive) tumour regions, and laser capture microdissection to harvest regions of interest. RNA was isolated from those regions and analyzed by RNA-Sequencing. GeneGo pathway analysis revealed that the ErbB and Wnt pathways are significantly upregulated in the p-Erk positive dataset. In order to validate the importance of these pathways, we treated cells derived from the same KRasG12D- and MYC-driven mouse tumours with the pan-ErbB-family inhibitor Neratinib and the WNT-inhibitor LGK974. Single treatment with either inhibitor suppressed cell propagation, migration and invasion into Matrigel, whereas combined treatment had a stronger effect on both characteristics. A panel of KRas mutant human lung adenocarcinoma cell lines were similarly sensitive to at least one inhibitor or to the combination of both. With KRas being downstream of ErbB family receptors and EGFR- and KRAS-mutations being mutually exclusive in NSCLC, the reliance on ErbB family signalling in KRas mutant cells was not expected. These results suggest that broad-specificity inhibitors of these proteins may be effective against a broader spectrum of NSCLC than hitherto anticipated. These results moreover indicate significant cooperation between the Ras and Wnt pathways that likewise may be exploited for therapy. Individual p-Erk associated genes that are also amplified or overexpressed in human NSCLC were selected for an in vitro siRNA screen. A significant number of these genes also correlate with decreased overall survival of NSCLC and in particular lung ADC patients. Screening of 3 KRas mutant human lung adenocarcinoma cell lines revealed that a considerable number of genes is important for cell viability of all tested cell lines. Also, knockdown of certain genes considerably suppressed cell migration in two efficiently migrating cell lines. These results suggest, that I have identified a list of genes that play an important role in KRas mutant lung adenocarcinoma.

Advances in DNA sequencing technologies, particularly the advent of next generation sequencing (NGS) platforms, have revolutionised the field of metagenomics and allowed great progress to be made in the way that microbial communities are analysed. However, the wealth of data now available thanks to these advancements has made the possibilities far more numerous than just the obvious applications, with a wide variety of novel and diverse studies conceivable. The technologies themselves have also created further areas for research as better methods of handling the, often overwhelming in quantity and misleading in content, data are sought. The analysis carried out in this thesis demonstrates the wide range of study possible stemming from two experiments involving the sequencing of meiofauna DNA. The first of these involves community analysis of marine benthic meiofauna with particular emphasis on diversity and distribution. The second experiment involves the sequencing of pooled nematode samples in order to investigate the effects of sample richness and species relatedness on the generation of chimeric reads in sequencing data. It is shown that the data generated from these two experiments can be used to help formulate an algorithm to simulate PCR and therefore assist the generation of realistic noisy NGS data. These data can, in turn, be used to generate a simulated in silico microbial community for analysis, the results of which reveal insights into the accuracy of chimera detection software and the reliability of metagenetic community analyses. Worryingly, these results suggest that findings from similar in vitro studies are not as reliable as originally perceived. The same experimental data may also be used to investigate interactions between meiofauna species based on the incidental presence of prey species highlighted from the sequencing of individual meiofauna organisms. It is shown that these data can be used to accurately predict a nematode’s feeding type without having to examine the organism directly. It is also shown that there is no correlation between this method of inferring interactions between species and other methods which have been used in the past. This suggests that the earlier methods are inadequate when used for the detection of feeding interactions.

The endangered population of Amur tigers (Panthera tigris altaica) in the Russian Far East (RFE) faces an increasing risk of extinction due to infection with canine distemper virus (CDV). Short-lasting CDV infections are unlikely to be maintained in small populations of species with limited connectivity like tigers, where viruses fade out as susceptible hosts are depleted. Multi-host pathogens can persist in more abundant host species that can act as reservoirs of infection for threatened populations. This study combines assessments of host demography, serology and viral phylogeny to establish the relative contribution of domestic dogs and small bodied wild mesocarnivores to the maintenance of CDV, and as sources of infection for tigers. No antibodies were detected among tigers sampled prior to 2000 (n=19), but were measured in 35.7% of tigers in subsequent years (n=56), with at least five discrete transmission events occurring in one well-monitored population. Viral sequences from three tigers and one Far Eastern leopard (P. pardus orientalis) aligned within the Arctic-like clade of CDV, and shared recent common ancestry with viruses from 22 other wild carnivores from the region. Extensive spatial mixing of wild carnivore lineages suggested long chains of transmission consistent with a maintenance population. The exposure of tigers following 2000 coincides with increases in sable (Martes zibellina) numbers and hunting pressure, which could lead to greater pathogen prevalence and potential for spill over from a wild reservoir. The ratio of humans to dogs in rural areas in the RFE are among the lowest in the world (1.73), but the overall number of dogs has been stable during the period of increased CDV exposure in tigers. The only CDV sequence obtained from dogs shared high identity with Asia-4 clade viruses from dogs in Thailand, and was distantly related to wildlife sequences from the RFE. Serum antibodies were detected in dogs in all 26 communities where households were surveyed, but seroprevalence was higher in remote, less densely populated areas, suggesting possible transmission from wildlife. Although the maintenance of CDV in Russian dogs remains unconfirmed, the strong support for a wildlife reservoir limits options for managing the impact of CDV on tiger populations. The high turnover of large and often inaccessible populations of mesocarnivores combines with limitations in vaccine safety, efficacy and delivery, to render the control of CDV in a wildlife reservoir untenable. Managing the impact of CDV on Amur tigers must therefore focus on restoring the size and integrity of remaining tiger populations to withstand future outbreaks. The safety and efficacy of vaccine products for tigers should also be investigated, for use in low coverage vaccination strategies that could enhance the long-term persistence of tiger populations.