The role of the E4 protein in the human papillomavirus (HPV) life cycle is an enigma even though it has varied effects on cell behaviour and organisation in overexpression studies. Full-length E4 proteins are derived from E1^E4 spliced RNA transcripts and E1^E4 proteins from diverse HPV types interact with serine-arginine (SR)-specific protein kinase SRPK1, that regulates diverse cellular functions including RNA splicing. This thesis has sought to address the hypothesis that E1^E4 alters SRPK1 activity and influences SRPK1 functions in the HPV life cycle. This study has uncovered the novel finding that E1^E4 protein of HPV1, but not HPV5, 16 and 18, is a potent inhibitor of SRPK1 activity in vitro and in vivo and inhibition is dependent upon E1^E4 binding to SRPK1. Whilst HPV1 E1^E4 inhibits SRPK1 phosphorylation of cellular (ASF/SF2, SRp20, SC35, 9G8 and SRp75) and viral (HPV E2) SR protein substrates, it has only weak effects on SR protein cellular localisation and on cellular and viral RNA splicing in minigene systems. Addition of the small molecule inhibitor of SRPK, SRPIN340 to organotypic raft cultures of HPV18 genome-containing keratinocytes enhances the morphological features of HPV viral replication suggesting that the HPV may modulate SRPK activity to facilitate the virus life cycle.

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.

Rheumatoid arthritis (RA) is an autoimmune disorder characterised by its varied and unpredictable origin, eventual destruction of cartilage and bone and the perpetual length of treatment involved. Even though significant developments have taken place over the past couple of decades in the treatment, the efficacy of drugs for RA is a major concern due to the side effects involved. Methotrexate (MTX) is currently used as first line treatment due to its ability to modify the rheumatic conditions so that disease progression can be prevented. However, at the same time prolonged exposure to MTX can lead to severe side effects such as lung fibrosis and hepatotoxicity. Recent research has focused on developing an alternative to MTX with similar efficacy but with reduced adverse effects. One such promising option is curcumin, an active compound extracted from Indian spice Turmeric. Various studies have indicated the synergistic properties exhibited by curcumin and its ability to modulate the underlying inflammatory pathways involved in RA. However, no previous studies have been reported with regards to using a combination of MTX and curcumin for the treatment of RA. A novel RP-HPLC stability indicating method was developed in order to establish the compatibility of the two compounds. The method was developed using Waters Reverse Phase (XBridgeTM Shield RP18 4.6x250 mm, 5 µM) column. A gradient system, consisting of two mobile phases with acetonitrile concentrations of 35% and 60% respectively, was designed to optimise the separation of the two compounds while taking into consideration the difference in hydrophobicity. The wavelengths for detection were 305 nm and 430 nm for MTX and curcumin, respectively. The retention time for MTX and curcumin was 4.8 ± 0.10 min and 12.3 ± 0.10 min, respectively. The Page 4 of 192 total run time of analysis was 25 minutes. The developed method was validated for parameters such as accuracy, precision, linearity, limit of detection and lower limit of quantification. The system was tested through intraday and interday repeatability and reproducibility. The method was used to analyse the MTX and curcumin under different stress conditions such as pH, UV radiation, temperature and humidity to establish their compatibility. The degradation products are successfully separated and therefore, the method can be effectively used as stability indicating method. Gene expression profiling was performed using HFLS-RA cells treated with MTX and curcumin separately and concurrently. The DNA microarray data identified 53 genes that were downregulated and 21 genes that were upregulated in all the treated samples using both stringent and non-stringent filtering. The total of 13 genes were selected based on the fold change obtained in the microarray data and their potential as therapeutic biomarkers based on previous research. The gene validation using qRTPCR confirmed the higher efficacy of curcumin in the inhibition of the proinflammatory genes such as ANGPTL7, CD248, CH25H, COL14A1, CXCL12, CYTL1, IFITM1 and IL7. Curcumin was found to also increase the expression levels of genes associated with anti-inflammatory roles, namely BCAR4, CD274, HSPA6, OTP and RELT. The increased gene expression in samples treated with both MTX and curcumin confirmed the possible synergistic activity which is encouraging, taking in to account the fact that these compounds are compatible according to the stability studies carried out and thus could be used in combination for the treatment of RA. This could improve the current treatment by reducing the severity of side-effects attributed to MTX while maintaining the efficacy of the treatment due to the ability of curcumin to modulate specific therapeutic biomarkers involved in the RA pathogenesis.