The development of a novel photon-by-photon Bayesian analysis for time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) data, and its application to both real experimental biological and synthetic data, is presented in this thesis. FLIM is an intensity-independent and sensitive optical technique for studying the cellular envi- ronment and can robustly exploit Fo¨rster Resonance Energy Transfer (FRET) to enable protein-protein interactions to be located within living or fixed cells. Careful analysis of fluorescence lifetime data, often comprising multi-exponential kinetics, is crucial to elucidating FRET via FLIM. The developed Bayesian analysis is demonstrated to offer more accurate fitting of data with lower photon counts, allowing greater acquisition speeds. As well as revealing infor- mation previously unobtainable, such as direct error estimates, fitting model probabilities, and instrument response extraction, the developed approach allows for future extensions which can exploit the full probability distribution. In a section of this work already pub- lished [1], Bayesian mono-exponential analysis was shown to offer robust estimation with greater precision at low total photon counts, estimating fluorescent lifetimes to a level of accuracy not obtained using other techniques. Bayesian mono-exponential parameter es- timates obtained with the developed Bayesian analysis are improved compared to those obtained using maximum likelihood, least squares, and the phasor data fitting approaches. In this work, Bayesian bi-exponential analysis based on an improved fully-analytic time- domain FLIM system model is shown to also offer improved decay parameter estimates. The developed analysis offers fluorescence decay model selection by exploiting the hierarchical nature of Bayesian analysis. This innovation enables the quantitative determi- nation of the likelihood of the data being due to mono- or bi-exponential decay processes, for example. Model selection applied to FLIM promises to simplify processing where the exact kinetics are not known. Finally, the determination of an approximated instrument response function from observed fluorescence decay data alone is also possible.

Nuclear envelope spectrin repeat proteins, or Nesprins, are a novel family of nuclear and cytoskeletal proteins with rapidly expanding roles as intracellular scaffolds and linkers. Nesprins are characterized by a central spectrin repeat (SR) rod domain and a C-terminal KASH domain, which acts as a nuclear envelope (NE) targeting motif. At the NE, via interactions with the Sun domain family of proteins and the nuclear lamina, nesprins on both the inner and outer nuclear membranes form the linker of the nucleoskeleton and cytoskeleton (LING) complex. This complex requires the giant nesprin-1 and nesprin-2 isoforms, which possess a pair of N-terminal calponin homology domains that bind directly to Filamentous-actin. However, via alternative promoter usage and alternative 3’ end processing, the nesprins are able to generate multiple mRNA transcripts, leading to the production of diverse tissue specific isoforms with potential roles beyond the NE. To explore further the capacity of nesprin-1 to generate alternative transcripts, 5’ and 3’ RACE was performed to identify cDNA ends which represent novel 5’ and 3’ untranslated regions (UTRs) respectively. By alternatively combining the differential 5’ and 3’ UTRs, multiple tissue specific nesprin-1 variants could be generated. Transfection of tagged constructs showed localizations to multiple sub-cellular compartments such as the nucleolus, focal adhesions, actin stress fibres and cytoplasmic particles, supporting the notion that nesprins are more than NE-cytoskeletal couplers. One of these novel nesprin-1 variants, p50Nesp1, was found to localize to cytoplasmic RNA granules called mRNA processing bodies (P-bodies). Using GST pull-downs and co-immunoprecipitations (co-IPs), p50Nesp1 was found to complex with mRNA decapping factors and translational repressers. More importantly, p50Nesp1 associated strongly with microtubules, both in vitro and in vivo, and was required for scaffolding P-body complexes to microtubules. By disrupting P-body-microtubule association with a dominant negative p50Nesp1 construct, time-lapse microscopy demonstrated impairment of fluorescently-labelled P-body proteins. Furthermore, this disruption resulted in P-bodies failing to associate with RNA stress granules and transferring p-globin mRNA reporter transcripts between compartments during the stress response. Further co-IP experiments identified a host of mRNA processing proteins that also associated with nesprin-1, including Matrin-3; an abundant nuclear matrix protein involved in a number of key nuclear processes. A novel isoform of matrin-3 localized to P-bodies and was required for miRNA-mediated translational repression. By identifying and tethering matrin-3 P-body localizing domains to a luciferase reporter construct, it was also found that matrin-3 could induce translational repression, which was significantly hampered when a single point mutation (S85C), previously described in a form of autosomal dominant distal myopathy, was introduced.

The phenotype of airway smooth muscle (ASM) differs in asthmatic patients compared to healthy individuals. Hyperplasia, hypertrophy, increased contraction and altered synthetic capabilities of asthmatic airway smooth muscle cells (ASMCs) have all been reported, however, the molecular mechanisms underlying a number of these changes remain unclear. This project utilizes molecular techniques to investigate the regulation of gene expression in cultured ASMCs isolated from both healthy and asthmatic individuals. microRNAs (miRNAs) are short, non-coding RNAs that exert a post-transcriptional regulation on gene expression by targeting specific mRNAs for down regulation through either mRNA degradation or translational repression. During this project microarray studies have been performed to determine differences in the miRNA expression profile of cultured ASMCs isolated from healthy or moderate asthmatic volunteers. These studies have identified a number miRNAs with differential expression in asthmatic ASMCs compared to healthy controls. Of particular note, it is demonstrated that the abundance of both mature miR-155 and its host transcript, BIC, are approximately three-fold lower in cultured asthmatic ASMCs compared to healthy controls. Various experimental approaches to identify endogenous targets for miR-155 in cultured ASMCs are described. The advent of next-generation sequencing technology has had a great influence on the field of chromatin and transcriptional regulation. In particular it has been shown that specific histone modifications demarcate functional and architectural regions within the genome. Investigations using chromatin-immunoprecipitation followed by next-generation sequencing (ChiP-seq) to generate a profile of histone methylation enrichments across the genome of cultured healthy ASMCs are described. It is envisaged that this data set will provide a resource for researchers interested in the transcriptional regulation of gene expression in ASM, in particular it will facilitate the identification of novel distal elements involved in the regulation of specific loci.

Retinoic acid (RA) is the main endogenous bioactive form of vitamin A that plays an important role in many biological events by regulating gene expression. One of the major mechanisms via which endogenous RA regulates gene expression is through the canonical signalling, which involves binding and activation of RA receptors (RARs) by RA. It is well-acknowledged that the canonical signalling of endogenous RA is indispensable for embryonic kidney development but its role in the kidney after birth is not as well-established. It is hypothesised that the canonical signalling of endogenous RA continues to regulate gene expression in the kidney after birth. In the kidney of young and adult RARE-hsp68-lacZ mice, a constitutive signal of RA response element (RARE) activation was observed. The signal was largely localised to collecting duct principal cells, inner medullary collecting duct cells, and intercalated cells. In concordance with the in vivo observation, basal RARE activity was detected in mIMCD-3 cells, a cell line derived from mouse inner medullary collecting ducts. The RARE activity was likely a result of constitutive activation of RARs given rise by cell-autonomous synthesis of endogenous RA as the activity was suppressed by antagonists of RARs and by an inhibitor of RA synthesising enzymes. Using mIMCD-3 cells as an in vitro model, target genes of endogenous RA/RARs were identified using microarray at the whole genome level, and the results were validated with reverse transcription quantitative polymerase chain reaction. Gene ontology analysis on the validated genes suggests their involvement in vitamin A metabolism, as well as in some classical and novel functions of collecting ducts including kidney development.

No description available.

RNA Polymerase II (RNAP II) performs transcription in the context of chromatin, which is the physiological substrate of all DNA-mediated processes. While several independent studies investigated the proteins associated with the high-salt soluble form of human RNAP II, the proteomic composition of the native chromatinassociated RNAP II was unknown. In this work, a procedure to purify native chromatinassociated RNAP II has been developed and the DNA helicase RECQL5 was identified as a novel RNAP II-associated protein. Reciprocal proteomic analysis of RECQL5- associated proteins revealed that RECQL5 co-purifies with RNAP II complex, as well as RNAP II elongation and initiation factors. Reconstitution of RNAP II transcription with highly purified transcription factors revealed that RECQL5 inhibits both the early initiation and processive elongation stages of RNAP II transcription. Moreover, RECQL5 can stimulate cleavage of nascent transcripts associated with stalled RNAP II elongation complexes. Importantly, all these effects require a stable RECQL5-RNAP II interaction. Genome-wide association analysis by using a ChIP-Seq approach revealed that RECQL5 is enriched in promoters and coding regions of several RNAP II transcription units in the human genome. These results collectively suggest that human RECQL5 protein is a bona fide RNAP II transcription factor and plays multiple roles in RNAP II transcription cycle. Finally, proteomic analyses of other chromatin-associated proteins suggest that the initial purification approach developed for RNAP II can be utilized as a generic strategy. Together, the observations presented in this work illustrate that the previous view of human RNAP II transcription machinery was far from complete, and analysis of native chromatin-associated proteins may expand the current knowledge about other DNA-mediated processes in human cells as well.

The research outlined in this thesis was primarily designed to study certain genes and their encoded proteins which regulate the development of the malaria parasite, <I>Plasmodium falciparum</I>, through its intra-erythrocytic life cycle. The work has involved molecular cloning and characterisation of a <I>P. falciparum</I> homologue of the <I>ras</I>-related nuclear GTP-binding protein, Ran/TC4. This protein has been shown to play a key role in control of the cell cycle in mammalian and yeast systems. In this work, the homologous <I>P. falciparum</I> gene, <I>pfran</I>, has been cloned and characterised. Its predicted protein product is 214 amino acids long and contains consensus motifs found in the Ras superfamily. The expression of a 1.5 kb <I>pfran</I> transcript has been found to change during the cell cycle, reaching a peak at the trophozoite and schizont stages. Western blotting and immunoprecipitation using a polyclonal antiserum raised against the expressed protein have shown that <I>pfran</I> encodes a protein 27 kDa in size. Immunofluorescence assays have shown that <I>pfran</I> is localised in both the nucleus and cytoplasm of the parasite. Some experiments were undertaken to express <I>pfran</I> in yeast which had only limited success. Ran/TC4 is proposed to function in conjunction with the chromatin-binding protein RCC1, (Regulator of the Chromosome Condensation -1), to couple the completion of DNA synthesis with initiation of mitosis. This takes place by the activation of a cyclin-p34<SUP> CDC2</SUP> complex. To understand better the regulation of the cell cycle in malaria parasites, and the role of Ran in this process, the <I>p. falciparum</I> homologue of the RCC1 has been cloned and partially sequenced. Expression of this gene and its possible association with <I>pfran</I> have been studied. The implications of these findings are discussed in the thesis.

The work presented in this thesis describes the development of, and investigation into a novel genetic method. Linkage Group Selection (LGS), which achieves rapid <i>de novo</i> location of genes encoding selectable phenotypes of malaria parasites. A phenotype-specific selection pressure is applied to the uncloned progeny of a genetic cross between two malaria parasites that differ in the phenotype under investigation. Selected and unselected progeny are analyzed using genome-wide quantitative genetic markers. Markers of the “sensitive” parent, which are reduced after selection, are sequenced and located in genomic databases. They are expected to be closely linked to gene(s) determining the phenotype under selection. The validation of LGS presented in this work was carried out with the rodent malaria parasite <i>Plasmodium chabaudi chabaudi</i> using a phenotype, pyrimethamine resistance, whose controlling gene, that encoding dihydrofolate reductase (<i>dhfr</i>), is known. The results show that molecular markers closely linked to <i>dhfr</i>, and only those linked to this gene, were reduced or removed by pyrimethamine treatment in accordance with the expectations of LGS. In addition, experiments investigating the “fitness cost” of pyrimethamine resistance are described. The recombinant progeny of a cross between drug-sensitive and drug-resistant parasites were grown in infections in mice, and the proportions of the sensitive and resistance alleles of <i>dhfr</i> were measured throughout the infection. It was found that parasites carrying the pyrimethamine resistance mutation at <i>dhfr</i> grew, more slowly than those carrying the sensitive allele, indicating this specific mutation for drug resistance carried a fitness cost to the parasite. This result may have important implications for the design of effective drug policies in malaria endemic areas.

Bub1 is a conserved protein kinase that plays an essential role in the spindle checkpoint. The role of the Bub1 kinase activity remains elusive, and it is controversial whether it is required for spindle checkpoint arrest. In this study we use budding yeast as a model system to analyse the role of the Bub1 kinase domain, using a <i>bub1ΔK </i>allele lacking the C-terminal kinase domain. We show that <i>bub1ΔK </i>cells are able to initiate and maintain a mitotic arrest induced by microtubule de-polymerising drugs and kinetochore defects. Despite this, these cells display significant sensitivity to microtubule drugs and we show that this is because they are unable to accurately recover from spindle damage and mis-segregate their chromosomes at high rates. This phenotype is reminiscent of <i>sgo1Δ </i>cells in mitosis, and we show that Bub1 kinase is required for accurate localization of Sgo1 to kinetochores in metaphase. We then demonstrate that both Bub1 kinase and Sgo1 are required for efficient chromosome bi-orientation, and we suggest that this is due to inability to detect a lack of tension at the kinetochores. Finally, we propose that Bub1 kinase targets Sgo1 protein to kinetochores to ensure efficient chromosome bi-orientation.

The specific locus mutagenesis test generated thirty overlapping deletions at the brown (<I>Tyrp</I>1) locus. This has permitted high resolution molecular and functional analysis of a 7-9cM interval on mouse chromosome 4. Complementation analysis revealed four new functional units in the central to distal part of this region, two of which are early embryonic lethals. This study has shown that one appears to result in neonatal death. The fourth, <I>baf</I> (brown associated fitness) has a sub-viable phenotype, demonstrating poor growth, possible gut abnormalities, nervous behaviour and death at around weaning age. This phenotype appears to be highly variable, with some homozygotes surviving to adulthood. A comparative study of the different deletions in their homozygous state has been undertaken to determine if these phenotypes or their severity are genetically separable. Generation of fine structure map of a 2.5cM region encompassing these genes containing over 30 markers has been completed. Techniques including sample sequencing, exon trapping, cDNA selection and homology mapping have identified transcripts from this region. This has resulted in a comparative transcript map of the mouse chromosome 4 interval and the conserved syntenic region on human chromosome 9. Several transcripts have been placed on this map and of particular interest is a transcript encoding 13 PDZ domains which maps distal to (<I>Tyrp1</I>). PDZ domains are known to interact with a number of proteins at specific cellular junctions and may be responsible for the sub-cellular localisation and clustering of proteins into functional complexes. There is evidence that the protein interacts with the C-terminus of the 5-HT2c receptor.