No description available.

Telomeres are DNA-protein complexes that help protecting the end of linear chromosomes. They consist of repetitive DNA, in mammals the repeat unit is the hexanucleotide TTAGGG, these repeats span 5-20 kb. Under normal conditions in somatic cells, telomeres get shorter with every population doubling until they reach a critical length and then, the cell enters a checkpoint called senescence or M1 where it stops dividing. If the cell escapes senescence and continues dividing with further telomere shortening, it reaches a second checkpoint called crisis or M2. Crisis is characterized by telomere dysfunction leading to genomic instability that can end with cell death. However, some cells achieve to maintain telomere length by activating a telomere maintenance mechanism (TMM). The presence of a TMM is a hallmark of cancer cells. Two TMM have been described in human cells, one is the through the enzyme telomerase, which is active in 85% of cancers, and the second is a homologous recombination (HR) based mechanism called Alternative Lengthening of Telomeres (ALT) active in 15% of cancers. The evidence that the ALT pathway relies in HR was the observation that sequences can be copied from one telomere to another in ALT+ but not in telomerase+ cells and that several genes involved in HR are necessary for ALT progression. The ALT pathway is not the only event involving HR at telomeres. It has been shown that the human herpesvirus 6 (HHV-6) can integrate into human telomeres. Interestingly, HHV-6 possesses perfect telomeric repeats within its genome. The proposed mechanism for integration if through HR between the telomeric repeats present in the virus with the human telomere repeats. The aim of this work is to unravel the molecular mechanism underlying the ALT pathway and HHV-6 integration. The data obtained will contribute to the understanding of HR in human telomeres.

Polyadenylation by the mitochondrial poly(A) polymerase (mtPAP) is a crucial step of post-transcriptional modification in mammalian gene expression. In human mitochondria, polyadenylation is required for completion of seven UAA stop codons following complete processing of the major polycistronic RNA unit. Patients homozygous for a 1432A>G mutation in the PAPD1 gene, which encodes mtPAP, suffer from symptoms consistent with mitochondrial disease including autosomal-recessive spastic ataxia and optic atrophy. The principal defect of the 1432A>G mutation is short adenylate tails on mt-mRNAs. Fibroblast lines from patients harboring the 1432A>G PAPD1 mutation were established, and analysis of mitochondrial gene expression showed non-uniform dysregulation. For mt-mRNAs and translation products, there is a mix of depletion, stabilization and no effect, leading to major deficits at steady-state protein levels and of respiratory complexes. To confirm the pathological nature of the mutation, a complementation experiment was performed, which showed that expression of the WT PAPD1 gene rescued the mutant phenotype. To assess whether catalytic activity was altered in the mutant enzyme, in vitro polyadenylation assays with WT and N478D recombinant mtPAP were undertaken. The N478D mtPAP was found to generate the short oligo(A) tails as observed in vivo. In addition, the presence of the LRPPRC/SLIRP complex increased the maximal poly(A) extensions generated by both WT and mutant mtPAP. Finally, experiments were undertaken to identify factors potential interacting with mtPAP. The major interacting factor was found to be ATAD3, a protein reported to be involved with multiple mitochondrial processes involving DNA and translation machinery in the form of nucleoids or mitoribosomes respectively. In summary, these investigations provide insights into the impact and regulation of mitochondrial polyadenylation, and contribute towards unraveling the complexities of post-transcriptional maturation in human mitochondrial gene expression.

The microscopic observation of thin sections of anatomical tissue provides knowledge about its molecular and cellular constituents, which is crucial in identifying pathologies, understanding the structure and function of internal organs and for the construction of anatomical atlases. The digitization of these sections yields two dimensional (2-D) images which provide rich anatomical and functional detail at both microscopic and macroscopic level. While the spatial resolution, contrast and specificity of these images continue to outperform the classic three dimensional (3-D) imaging modalities, such as magnetic resonance imaging, their quality and, crucially, quantitative analysis is still limited. The reason is that the organs or anatomical structures of interest are inherently 3-D objects and the analysis of their shape, the computation of their volume, or the comparison of their characteristics across individuals cannot be accurately performed on the basis of 2-D sections alone. Therefore, 3-D volume reconstruction from 2-D histological images usually constitutes a first step in the morphological analysis of the structures imaged by histology. Yet, the loss of 3-D spatial alignment together with the numerous artefacts occurring in the 2-D image acquisition process make reconstruction a difficult task. The work presented in this thesis is based on the observation that the quality of reconstructed histological volumes is usually assessed by considering the smoothness of some reconstructed structures of interest. Our research has two novel contributions: (1) two 3-D reconstruction methods for 2-D histological images which use smoothness as a means to drive the reconstruction process itself; (2) a quantitative measure of smoothness to assess the quality of reconstructed volumes. We apply the reconstruction techniques to various datasets of both synthetic and real histological images. The qualitative visual inspection of the reconstructed volumes is complemented with the quantitative measurements of smoothness with excellent agreement. We also perform a robustness analysis of the proposed reconstruction methods where we evaluate their behaviour in the presence of a variety of geometrical perturbations and typical histological artefacts.gg

Intake of the micronutrient selenium, which is incorporated into selenoproteins in humans, has been implicated in affecting risk of colorectal cancer. A genetic variant in the gene encoding the selenoprotein glutathione peroxidase 4 (GPx4) has been reported to influence colorectal cancer risk. In this study the role of GPx4 was investigated in the Caco-2 intestinal cell line using RNA silencing. GPX4 expression was knocked–down by ~60% and an unbiased gene microarray analysis of the total Caco-2 cell transcriptome was carried out using Illumina HumanHT-12v3 beadchips. The data were validated by real-time PCR. Ingenuity Pathway analysis showed that the major canonical pathways affected by GPX4 knock-down were oxidative phosphorylation, ubiquinone biosynthesis and mitochondrial dysfunction and the top two toxicological lists were mitochondrial dysfunction and oxidative stress. Western blotting and real-time PCR confirmed that knock-down affected target genes encoding components of respiratory complexes I, IV and V as well as the protein apoptosis-inducing factor (AIF). GPX4 knock-down increased levels of mitochondrial reactive oxygen species and oxidised lipid, and decreased mitochondrial adenosine triphosphate (ATP) levels and mitochondrial membrane potential. Time course experiments showed changes in AIF expression preceded those in the respiratory complexes. GPX4 knock-down increased apoptosis and changed protein expression of Caspase-9, Bax and Bcl-2. Treatment of cells with the antioxidant mitoquinone prevented the effects of GPX4 knockdown on mitochondrial reactive oxygen species, oxidised lipid and mitochondrial membrane potential but not the effect on AIF. These data suggest that in intestinal epithelial cells GPx4, through effects on lipid peroxidation and AIF, plays a complex role in maintaining the oxidative phosphorylation system and protecting mitochondria from oxidative damage and apoptosis.

No description available.

A reduction in cell volume is a fundamental feature of apoptosis. This thesis has characterised changes in cell volume, together with nuclear changes, occurring in several models of apoptosis including, murine L1210 B-cells treated with mechlorethamine and human CEM-C7 A lymphoblastoid cells treated with dexamethasone, etoposide or subjected to serum deprivation. In the etoposide and glucocorticoid apoptotic models, cell volume was measured by electronic cell sizing and flow cytometry and two distinct phases were observed. In the dexamethasone model, the first phase of cell shrinkage began 12 hours after the addition of dexamethasone (5 ILM) and progressed until 36 hours when chromatin condensation was detected in intact cells. Removal of dexamethasone before 36 hours (the pre-commitment period), resulted in reversal of the volume decrease and prevented the appearance of nuclear changes. In the etoposide model, cells initially swelled, within the first 8 hours, and then rapidly lost volume at the later time points. Multiparameter flow cytometry indicated that cell shrinkage and chromatin condensation were occurring simultaneously in individual cells. The presence of the anti-apoptotic protein Bcl-2 abrogated chromatin condensation without affecting cell shrinkage or cytosolic acidification. Partial inhibition of protein synthesis also delayed chromatin condensation without affecting cell shrinkage. Possible mechanisms behind the first and second phase of cell shrinkage, in the dexamethasone model, were investigated. In the first phase, there was a net loss of potassium but no change in cellular buoyant density or potassium efflux. There were no significant differences in the rates of volume recovery after either hypertonic or hypotonic stimuli. These observations favour a mechanism of cell shrinkage involving loss of the entire cytoplasmic contents, possibly following proteolysis, rather than loss of only osmolytes and water. The second phase of volume loss was coincident with chromatin condensation and was associated with cellular fragmentation and a reduction in cellular density. In conclusion, the exact relationship of apoptotic cell volume changes to chromatin condensation appears to vary from cell model to cell model with cell volume loss being mediated by multiple mechanisms which are both dependent and independent of cellular fragmentation.

The calcium-dependent antibiotic is a lipopeptide antibiotic produced by S. coelicolor A3 (2). Its structure comprises a cyclic undecapeptide containing a number of non-proteinogenic amino acids and an N- terminal 2,3-epoxyhenanoyl side-chain. A number of natural CDA variants have been characterised which arise by modification of specific amino acid residues within the peptide. The CDA biosynthetic gene cluster has been fully sequenced as a consequence of the S. coelicolor Genome Sequencing Project at the Sanger Centre, which was completed in 2002. The CDA biosynthetic gene cluster contains open reading frames encoding three non-ribosomal peptide synthetase (NRPS) genes (cdaPS1-3), fatty acid biosynthesis genes and a number of putative genes encoding enzymes for amino precursor biosynthesis and tailoring of the peptide. cdaPS1-3 appear to have a modular structure typical of linear NRPS genes where each module is responsible for recruitment of a specific amino acid into the peptide. It has been hypothesised that a domain of unknown function at the start of module 1 is responsible for the transfer of the 2,3-epoxyhexanoyl side-chain to the undecapeptide. Module 1 was the focus of the investigation since this module is believed to have an initiating role in CDA biosynthesis. It was concluded from in vitro studies with overproduced module 1 protein from E. cola and S. lividans strains that module 1 adenylation domain is specific for the antibiotic serine, however attachment of serine to the PCP domain of module 1 was not demonstrated. A conserved serine residue in the PCP domain of module 1 was shown to be essential for binding of the 4'-PP cofactor by site directed mutagenesis of the PCP domain DNA: when this sequence was changed to encode alanine CDA production was abolished. A number of substrates were proposed for feeding to PCP domain mutants to re-establish CDA biosynthesis and this work is ongoing. The enzyme responsible for hydroxylation of asparagine at position 9 in CDA was investigated. This enzyme was thought to be a member of the α-ketoglutarate dependent Fe(II) di-oxidase family of enzymes because of the homology shared by the putative asparagine oxygenase (SC03236; asnO) and clavaminic acid synthase (CAS). An inframe deletion of SC03236 was introduced into the chromosome of S. coelicolor strains and analysis of secondary metabolites produced by the mutants suggested that CDA containing asparagine rather than hydroxyasparagine at position 9 was produced. It was concluded therefore that SC03236 was the gene encoding asnO. This was confirmed by complementation of deletion mutants with vector borne SC03236, which indicated a reversion to production of wild type CDA containing hydroxyasparagine by the complemented strains. Over-production of AsnO protein in E. coli was carried out and the protein used in hydroxylation assays with a number of different substrates and the required co-factors for the presumed α-ketoglutarate dependent Fe(II) di-oxidase. However, these experiments proved inconclusive and work in this area is ongoing.

No description available.

The bHLH transcription factor Hand1 is essential for placentation and cardiac morphogenesis in the developing embryo. However, how the activity of Hand1 is regulated in either lineage remains largely unknown. Here we demonstrate that Hand1 is anchored in the nucleolus and negatively-regulated by the murine orthologue of the human I-mfa domain-containing protein (HIC). Nucleolar sequestration controls Hand1 activity during the differentiation of rat choriocarcinoma-1 (Rcho-1) trophoblast cells. Hand1 is sequestered in the nucleoli of Rcho-1 stem cells but is released into the nucleoplasm at the onset of their differentiation into trophoblast giant cells. Site-specific phosphorylation of Hand1 was previously shown to modulate the affinity of Hand1 for its nucleoplasm E-factor binding partners. We demonstrate that Hand1 phosphorylation is required for its nucleolar release, as a pre-requisite for dimerisation and biological function. Moreover, the polo-like kinase Plk4 (Sak) is responsible for this phosphorylation event. Plk4 localises to the nucleolus of Rcho-1 stem cells at phase G2 and interacts with Hand1 in vitro and in vivo to promote mitotic cell cycle exit and entry into the endocycle. We also demonstrate that the B568 subunit of the PP2A phosphatase, shown previously to target Hand1 for dephosphorylation, is exported from the nucleus during Rcho-1 differentiation. In this thesis we present findings that describe a novel mode of Hand1 regulation that is a crucial step in trophoblast stem cell differentiation and placentation and support previous studies that implicate the nucleolus as a molecular 'sink'. We suggest that nucleolar sequestration is an important mode of protein regulation and this may impact on a broad range of transcription factors.