DNA sequencing technologies have developed quickly in the last decade, and new methodologies have moved into clinical practice. These can been used to investigate genetic causes of neonatal cholestasis. Neonatal cholestasis can be life-threatening and has a varied etiology. In chapter 3, a targeted next generation sequencing (tNGS) assay was designed and assessed for suitability for detection of known mutations in genes associated with cholestasis. In chapter 4, this was used to screen over 200 infants presenting with liver disease for mutations in the (ATP8B1), (ABCB11), (ABCB4), (NPC1), (NPC2) and (SLC25A13) genes. Diagnoses were made in 9% and single heterozygous mutations were in 9% of cases. In chapter 5, patients suspected of PFIC-related disease werE tested for mutations in the (ATP8B1), (ABCB11) and (ABCB4) genes. This study uncovered 27 novel sequence variants, including 22 in UK patients, expanding the known mutation spectrum of these disorders. In chapter 6, patients suspected of NPC and were tested for mutations in (NPC1) and (NPC2), or (SLC25A13), respectively. These studies have identified 134 novel NPC mutations and 4 novel CD mutations. Current and future DNA sequencing methods are discussed, as are new diagnostic strategies for genetically heterogeneous conditions like infantile liver disease.

Morpho-physiological characterization and whole genome transcript profiling of rice genotypes that belongs to sub-species Indica, Japonica and wild relatives were carried out under salt stress. The existence of qualitatively different mechanisms of salt tolerance across the genotypes was identified. Multivariate analysis was applied to categorize the genotypes according to their level of tolerance. Modified SAM analysis elucidated the trait specific expression of genome wide transcripts. Gene ontology enrichment analysis identified the genes involved in different molecular functions such as signal transduction, transcription factor and ion homeostasis etc. Gene network analysis identified the regulatory network of genes that are active in different tissues. The differential expression of transcripts of four tolerant and two susceptible Indica genotypes under stress were further analysed. The candidate genes for different biological processes and molecular functions are identified and discussed. Highly induced stimulus responsive gene Os01g0159600 (OsLEA1a) and Os05g0382200 (Nhx) can be mentioned for instance. The differentially expressed genes that are located within the salt stress related QTLs were also identified. The transcriptomics data were also used to predict the salinity tolerance of genotypes using OSC-PLSDA model. The combined physiological and transcriptomic approach of this study gives a complementary whole organism assessment of plants responses to salt stress.

Microbiological research has uncovered the basis of fermentation, infectious disease, vaccination and antibiotics. Now, a technological revolution leveraging DNA, the code of life, has allowed us to unravel cellular and evolutionary processes in exquisite detail. Today our need for new innovation is still great. The modern world is a challenging environment: over-population, climate change and highly mobile populations create a high risk of pandemic disease especially from viruses and many bacteria are now resistant to our life saving antibiotic drugs due to overuse. In hospitals, the spread of pathogens can be rapid and life threatening. Whole-genome sequencing has the power to identify the source of infections and determine whether clusters of cases belong to an outbreak. Portable, real-time nanopore sequencing enables sequencing to be performed near the patient, even in resource-limited settings. Integrating with existing datasets allows digital surveillance able to detect outbreaks earlier while they can still be contained. Early demonstrations of the power of whole-genome sequencing for outbreak surveillance have made it an area of intense interest and further development in laboratory methods and infrastructure will make it an important tool that can be deployed in response to future outbreaks.

Arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome is a severe multisystem disorder caused by mutations in VPS33B and VIPAR. A splice site mutation, VPS33B c.1225+5 G>C, was identified in a patient with attenuated ARC phenotype. Modelling of this mutation and missense mutations indicated that VPS33B-VIPAR interaction and co-localisation at recycling-endosome like structures is necessary for their function. Development of a murine ARC model began with ubiquitous removal of Vps33b, and in parallel, its homologue Vps33a. Both resulted in embryonic lethality between E7.5 and E8.5. A good ARC phenocopy was obtained the liver specific Vps33b knockout, Vps33b\(^{fl/fl}\)- AlpfCre. Bile acid levels were increased in comparison to control and the apical proteins CEA and BSEP were mislocalised, with the distribution similar to that in ARC livers. In contrast, a Vps33b\(^{fl/fl}\)-Pf4Cre mouse did not result in a good ARC platelet phenocopy, with α-granules found present. They did however present with a mild platelet phenotype, with defects in stable aggregate formation under shear conditions and deficiencies in the α- granule proteins VWF and PF4, giving possible insights into the bleeding diathesis in patients. In conclusion, this work has produced suitable ARC models for further study of Vps33b function and provides a platform for the future development of gene therapy and drug treatments.

The production of retroviral vectors for gene therapy applications faces a number of challenges. Of primary concern is the low titre of vector stocks produced by packaging cells in culture and the inherent instability of retroviral vector activity. A systematic investigation of culture parameters that can effect vector titre was conducted. Physical and chemical factors including temperature, pH, medium composition, dissolved oxygen and serum concentration were all assessed. In addition, a number of studies were undertaken to assess the effects of packaging cell growth rate on vector production. The use of a packed bed, as a novel system for large-scale vector production, was also investigated. Prolonged production of retroviral vector stocks was demonstrated in the packed bed system with immobilised packaging cells. Determination of the critical culture parameters allowed optimisation of culture conditions, which can be continuously controlled in the packed bed system, thereby ensuring optimal vector production throughout the production period. Furthermore, vector decay rates can be reduced by the immediate collection of vectors into a recovery vessel. The studies within this thesis will aid the development of appropriate procedures for the large-scale production and handling of retroviral vector stocks for human gene therapy applications.

Familial renal cell carcinoma (RCC) only accounts for 3% of all RCC, yet the study of these inherited forms has provided important insights into the more common sporadic RCC. Somatic VHL inactivation is found in 70% of sporadic clear cell RCC (ccRCC) though is rarely found in other forms of RCC including papillary and chromophobe types. VHL-independent RCC tumourigenesis is poorly understood and current research involves identifying novel RCC candidate genes to further understand the mechanisms involved. In this study a constitutional balanced translocation, t(5;19)(p15.3;q12), associated with familial RCC was characterised using an oligonuleotide CGH array followed by genomic sequencing and the previously uncharacterised gene, UBE2QL1, was found to be disrupted by the 5p15.3 breakpoint. UBE2QL1 expression was down-regulated in 78.6% of sporadic RCC and UBE2QL1 promoter region hypermethylation and gene deletions were detected in 20.3% and 17.3% of sporadic RCC, respectively. Re-expression of UBE2QL1 in deficient RCC cell lines suppressed anchorage independent growth and colony formation. UBE2QL1 shows homology to the E2 class of ubiquitin conjugating enzymes and was shown to possess an active-site cysteine (C88) that is monoubiquitinated in vivo. In addition, UBE2QL1 co-immunoprecipitation and co-localisation studies demonstrated a protein interaction with FBXW7 (an F box protein for the SCF E3 ubiquitin ligase) and was shown to facilitate the degradation of the known FBXW7 substrates, cyclin E1 and mTOR. These findings demonstrate that UBE2QL1 functions as a novel renal tumour suppressor gene and ubiquitin conjugating enzyme.

Hepatitis C virus (HCV) is a global health problem with over 150 million individuals infected worldwide. Many of these patients will develop end-stage liver diseases, such as cirrhosis and hepatocellular carcinoma (HCC), and will require liver transplantation. HCV exists as a heterogeneous population in infected individuals, however, the processes which maintain this genetic complexity are unknown. Recent observations suggest that HCV transmits between hepatocytes via a cell-to-cell route of infection, supporting a “demic” model of evolution where HCV diversity arises from independent evolution in small isolated hepatic populations. To investigate the distribution of HCV within the liver we sampled eight segments of the liver explant from 22 HCV infected subjects undergoing liver transplant and measured viral RNA burden and sequence diversity. Comparable HCV RNA levels were observed across all 8 samples from a single liver, however, between patients we observed a 100-fold range in the hepatic viral load that was independent of hepatic expression of anti-viral and pro-viral interferon stimulated genes (ISGs). Sequence analysis of the viral envelope E1E2 region, obtained from PCR generated single molecules or ultra-deep sequencing approaches, showed minimal evidence of genetic compartmentalisation between hepatic sites or between the liver and plasma. Modelling the HCV population structure in infected patients will have a major impact on our understanding of how HCV escapes host immune responses and anti-viral therapies.

Biosynthetic steps in the mupirocin (pseudomonic acids) biosynthetic pathway of Pseudomonas fluorescens NCIMB 10586 have already been deduced. Putative functions of most of the genes of \(mup\) cluster have been assigned although exact sequence of steps in the pathway and their timings are not yet known. Thiomarinols are another group of anti-bacterials produced by Pseudoalteromonas sp. SANK 73390. Very little is known about the biosynthesis of thiomarinols that share striking structural similarity with pseudomonic acids in their polyketide and fatty acid moieties. This similarity is reflected at genetic level as significant similarity in amino acid identity between the products of at least 27 ORFs in these biosynthetic clusters. This project aimed to learn more of biosynthetic steps in the biosynthesis of mupirocin and thiomarinol antibiotics by testing for functional cross-complementation between pair of genes or a group of genes whose products show significant homology. Surprisingly, only two genes \(tmlJ\) and \(tmlS\) out of nine studied showed complementation in the \(mup\) system. Findings suggested protein-protein interactions limited interchangeability of equivalent functions between two biosynthetic systems. It was shown by expressing related genes as groups for complementation in the \(mup\) system that it was possible to confirm specificities of such interactions.

DNA associates with proteins to form chromatin which is essential for the compaction of the DNA into the cell nucleus and is highly dynamic in order to allow the different biological processes of the DNA to occur. Chromatin compaction is achieved at different hierarchical levels: the 10nm fibre (DNA associates to nucleosomes formed by different histones), the Higher Order Chromatin fibre and the 300 nm chromosome structures. This study has shown that both H1 and H4 histones play a crucial role in preserving meiotic as well as mitotic chromosome structure and functional genome integrity in Arabidopsis. The role of the different linker histone H1 isoforms as well as the core histone H4 in Arabidopsis thaliana was investigated using T-DNA and RNAi mutant lines which showed different meiotic defects. Chromosomal breaks as well as non-homologous connections in the h4RNAi were linked to 45S/5S rDNA disorganisation, suggesting that H4 preserves chromosome integrity at these rDNA regions. Ath1.1 mutant presented univalents and reduced chiasma frequency at metaphase I, linked to a severe defect in ASY1 localisation on the meiotic chromosome axes. Thus, indicating that histone H1.1 is vital for proper chromatin axis organization that permit normal loading of recombination machinery proteins in Arabidopsis.

Parkinson’s disease (PD) is a neurodegenerative disease which causes tremor, muscular rigidity and bradykinesia (slowness of initiation of voluntary movement with progressive reduction in speed and amplitude of repetitive actions). Although the underlying causes remain unknown, there is evidence that genetic factors play an important role in the disease process. In this thesis I investigated the role of a recently identified hereditary PD gene leucine rich repeat kinase 2 (LRRK2) in PD in the United Kingdom (UK). In this thesis I have confirmed the importance of pathogenic LRRK2 mutations in UK familial PD (fPD). In addition I identified three novel frameshift mutations. I investigated the functional effects of two of these mutations and provide evidence that nonsense mediated decay (NMD) is occurring in LRRK2-PD. In this thesis I also present data from an extensive screen of LRRK2 in UK subjects with sporadic PD (sPD). This confirms the importance of pathogenic LRRK2 mutations in UK sPD. In addition I report a novel missense mutation in the GTP cyclohydrolase I gene (GCH1) in a kindred with phenotypes ranging from PD to Dopa-responsive dystonia. The association of this novel GCH1 mutation with late onset parkinsonism suggests a potential role for GCH1 in PD.