Mammalian genomes contain huge numbers of short interspersed elements (SINEs). An extreme case is provided by the human genome, which carries ~106 copies of Alu SINEs that together account for ~10% of total chromosomal DNA. SINEs spread by retrotransposition, which depends on their transcription by pol III. This transcription is heavily suppressed. Silencing is thought to involve DNA methylation and packaging the SINEs into chromatin structures that deny access of transcription factors. It has been argued that this may be of great importance to prevent SINEs from competing with essential genes for a limited pool of transcription machinery. Our investigation of this has revealed some unexpected findings. This study has also investigated the effects of SWI/SNF chromatin remodellers on tRNA transcription.

Pyrimidine uptake has previously been investigated in Trypanosoma brucei procyclics and partly investigated in promastigotes of Leishmania major; however, no such study has been performed using bloodstream forms of Trypanosoma or promastigotes of Leishmania. Here we report a comprehensive study of pyrimidine salvage and metabolism in bloodstream forms of Trypanosoma and promastigotes of Leishmania species. In T. b. brucei bloodstream forms, the uptake of 3H-uracil and 3H-tymidine each appeared to be mediated by a single transporter, designated TbU3 and TbT1, respectively. The procyclic uracil transporter,TbU1, has a high affinity for uracil, with a Km value of 0.46 ± 0.09 μM and Vmax of 0.65 ± 0.008 pmol (107cell)-1 s-1. These values were similar for TbU3 (Km = 0.54 ± 0.11 µM; Vmax = 0.14 ± 0.03), but the main differences between TbU1 and TbU3 are their sensitivity to uridine and 4-thiouracil. Thymidine uptake is detectable at 10 μM over a period from 5 to 30 minutes. This uptake was not inhibited by uracil which indicates that TbT1 is a novel thymidine transporter. The uptake of other pyrimidines, including uridine and 2’-deoxyuridine, by BSF are investigated here but these substrates were also transported by TbU3, and no additional pyrimidine transport activities were found. In L. mexicana and L. major, the uptake of 3H-uracil and 3H-uridine was mediated by separate transporters, designated as follows; for uracil uptake LmexU1, LmajU1; and for uridine uptake LmexNT1, LmajNT1 and LmajNT2, respectively. LmexU1 is a uracil transporter with high affinity to uridine and 2’deoxyuridine, and the LmexNT1 is a nucleoside transporter with broad specificity for purine and pyrimidine nucleosides. L. major uracil transporter (LmajU1) has already been reported by others; and here we report that there are also two distinct uridine transporters expressed in L. major. LmajNT1 is a high affinity uridine transporter which is also inhibited by uracil, inosine and adenine; LmajNT2 is low affinity uridine transporter, with very poor affinity for uracil, inosine and adenine. However, both transporters are inhibited by 2’-deoxyuridine, thymidine and adenosine. Several fluorinated pyrimidine analogues were assessed against kinetoplastid cells, the most effective compounds, which displayed EC50 values at micromolar level, are 5-FU, 5F-2’dUrd, 5-FOA (only against T. brucei BSF) and 5F-Urd (only against L. major). We induced resistance to 5-FU, 5-F2’dUrd and 5-FOA by in vitro exposure of Tbb-BSF and promastigotes of L. mexicana and L. major. The resistance was performed by stepwise increase concentration of the drugs. For T. b. brucei BSF, the resistance factors of clonal lines were 131, 825, and 83-fold, respectively. For L. mexicana and L. major, the resistance factor for 5-FU were 147 and 17-fold, and for 5F-2’dUrd were >3500 and 381-fold, respectively. We also measured 3H–pyrimidine uptake in these cell lines; the resistant bloodstream form strains showed no changes in pyrimidine uptake, with one exception, which is a 76% reduction in 5-FU uptake. In contrast, each resistant strain of Leishmania spp had lost its natural pyrimidine transporter. For example, Leishmania cells resistant to 5-FU had lost uracil transport activity, and cells that were resistant to 5F-2’dUrd had lost uridine transport activity. In addition, we identified kinetoplastid genes that appeared to be associated with resistance to fluorinated pyrimidines. Based on these findings, metabolomic analysis of fluorinated pyrimidines in T. b. brucei resistant cell lines was performed in comparison with parental wild-type; for Leishmania species we only investigated the metabolism of fluorinated pyrimidine in wild type cells, as the fluorinated analogues were simply not taken up in the resistant clones. The metabolomic analysis data showed that, in T. b. brucei, 5-fluorouracil and 5-fluoro orotate are incorporated into a large number of metabolites, but likely act through incorporation into RNA. 5F-2’dUrd and 5F-2’dCtd are not incorporated into nucleic acids but act as prodrugs by inhibiting thymidylate synthase after conversion to 5F-dUMP. Cells treated with 5-fluoro-2’deoxyuridine showed an increase of dUMP, which suggest a block in thymidylate synthase or possibly thymidylate kinase. We also present the most complete model of pyrimidine salvage in T. brucei to date, supported by genome-wide profiling of the predicted pyrimidine biosynthesis and conversion enzymes. The effect of fluorinated pyrimidine analogues in the two Leishmania species was almost identical. Each of the tested drugs (5-FU, 5F-2’dUrd and 5F-Urd) produced a limited number of fluorinated metabolites, and their common mode of action was inhibition in thymidylate synthase by 5F-dUMP and thymidine kinase by 5F-2’dUrd. Interestingly, we found that the cause of L. mexicana resistance to 5F-Urd was due to the absence of the 5F-2’dUrd metabolite, as a result of the rapid conversion of 5F-2’dUrd to 5F-dUMP; also we suggest that, in L. mexicana, but not in L. major the high affinity salvage of thymidine is sufficient to provide the cells with thymidine deoxynucleotides. It has been found that pyrimidine salvage is not an essential function for Leishmania cells in vitro conditions. However, it is not known whether either, pyrimidine salvage or biosynthesis, or both of these systems are essential to the trypanosomes in vitro and in vivo study. As T. b. brucei bloodstream forms grew unimpeded in vitro in the complete absence of pyrimidines, uptake is clearly not essential. Disruption of the pyrimidine biosynthesis pathway by deletion of the OMPDCase/OPRTase gene resulted in pyrimidine auxotrophic trypanosomes that were unable to grow in the absence of added pyrimidines. The phenotype was rescued by addition of uracil, and to a lesser extent by some pyrimidine nucleosides. Pyrimidine starvation led rapidly to DNA fragmentation. Adaptations to low pyrimidine availability included upregulation of uracil transport capacity and of uridine phosphorylase expression. However, pyrimidine auxotrophic T. brucei were able to establish a high parasitemia in mice. We therefore conclude that pyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity and strongly increased sensitivity to fluorinated pyrimidines. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the pyrimidine biosynthesis pathway is not a viable drug target, although any interruption of pyrimidine supply was lethal.

Cellular growth is the accumulation of mass by a cell. This is required in order to ensure the maintenance of cell size upon cell division. As the majority of the dry mass of a cell is protein, protein synthesis is essential for cellular growth. RNA Polymerase III (Pol III) is crucial for protein synthesis as it transcribes the genes encoding 5S ribosomal RNA and the tRNAs – key components of the translation machinery. As a consequence, Pol III plays an important role on cell growth and proliferation. Pol III-mediated transcription is highly regulated. Cellular stress usually results in decreased Pol III-mediated transcription, while increased transcription occurs following exposure of cells to mitogens. Understanding how Pol III is regulated is particularly important as loss of regulation is associated with cancer, where Pol III-associated transcription factors and Pol III products are frequently found highly expressed. In this thesis, the role of p53, AP-1 and c-Jun N-terminal kinases (JNKs) in the regulation of Pol III-mediated transcription in humans is examined. p53 is induced in response to stress and modulates the expression of a vast array of target genes. p53 represses Pol III-mediated transcription by binding to the Pol III-specific transcription factor TFIIIB and inhibiting polymerase recruitment to Pol III-target genes. In Chapter Three, it is demonstrated that repression of Pol III-mediated transcription is not the universal response to p53 upregulation. Treatment with the chemotherapeutic drug doxorubicin induces p53 robustly, and results in rapid reduction in tRNA levels but this is not dependent upon the presence of p53. Indeed, it is demonstrated that stress does not always result in the repression of Pol III-mediated transcription. Exposure of cells to ultraviolet light leads to an increase in tRNA levels. This demonstrates the complexity of the regulation of Pol III-mediated transcription in response to stress. Together, c-Jun and c-Fos produce one form of the transcription factor AP-1. AP-1 mediates cell proliferation and cell death in response to mitogenic stimuli and stress via the binding and activation of target genes. c-Jun and c-Fos have been found binding Pol III target genes. In Chapter Four, their occupancy at tRNA genes is examined in detail and found to be associated with active genes. While AP-1 binding motifs are found at a subset of tRNA genes, this is not found to be associated with c-Jun or c-Fos occupancy. Instead, recruitment to tRNA genes may occur via the association with Pol III-specific transcription factors that is observed. JNKs can regulate Pol III-mediated transcription by altering the level of the Pol III-specific transcription factor TFIIIB. JNKs also phosphorylate and activate c-Jun and other transcription factors, such as STAT1 and ATF2, found at Pol III target genes. The role of these transcription factors at tRNA genes has not been determined. However, in Chapter Five, it is demonstrated that JNKs may regulate Pol III-mediated transcription independently of regulating TFIIIB levels. Treatment of U2OS cells with a JNK inhibitor results in rapid reduction of tRNA levels resulting from reduced expression of tRNA genes. However, the levels of TFIIIB subunits TBP and Brf1 are unaffected. It is examined whether this effect may occur through a JNK target at tRNA genes or via another possible route, such as the phosphorylation of the Pol III machinery by JNK. Data in Chapters Four and Five suggest c-Jun and JNK may positively regulate Pol III-mediated transcription. This is consistent with their activation by mitogens, as more Pol III products are required for increased cell growth and proliferation. In conclusion, the data highlights the many pathways that converge to regulate Pol III-mediated transcription, and the complexity that arises as a consequence of this.

Cells communicate with each other and respond to environmental cues by sending and receiving signals. Many external signals (ligands) are detected through G protein-coupled receptors (GPCRs), a major class of transmembrane proteins. GPCRs transduce these external signals into appropriate intracellular responses, enabling the cell to adapt to its environment. Malfunctions in these signalling pathways can lead to a range of human diseases and hence GPCRs have become attractive candidates for pharmacological design. The activation of a single receptor has the ability to induce numerous intracellular responses. Coupling this with the great number of different GPCR-types expressed in human cells means that understanding the basic principles of signal transduction and termination in humans is complicated. This study utilises the more simplistic eukaryotic yeast Schizosaccharomyces pombe (S. pombe) to overcome this complexity, as it contains only two GPCR types and hence the cross-talk between pathways is greatly reduced, whilst the structure and signalling functions of GPCRs are often evolutionarily conserved between yeast and humans. Mathematical modelling was used to aid the understanding of GPCR signalling in S. pombe and to inform experimental design. Speci�cally, an ordinary differential equation model �rst developed by Croft et al. (2013) was extended to include all known downstream signal transduction, regulation and termination events. This model is the �rst of its kind to describe a whole GPCR signalling pathway within S. pombe. Although it accurately predicts the cellular response to GPCR signalling it could only reproduce the biological plateau in temporal response with the addition of a 'yet unknown mechanism' GPCR degradation term. This motivated the investigation of how GPCRs in S. pombe are internalised from the plasma membrane in response to ligand stimulation. The primary mechanism for signal termination is via internalisation of the GPCR. This study identi�ed three potential casein kinases (Cki1, Cki2 and Cki3) that promote internalisation of the S. pombe GPCR Mam2. Microscopy analyses in combination with quantitative transcriptional, cell growth and cell cycle position assays uncovered a novel role for these kinases: that Cki2 regulates cell size during vegetative growth, Cki1 and Cki3 regulate the GPCR-response pathway and that Cki3 is essential for completing cytokinesis in S. pombe that have already undergone formation of a conjugation tube in response to ligand. Confocal microscopy of uorescent labelled Mam2 indicated a role for Cki2 in the internalisation and hence termination of the GPCR-response pathway. These findings add to the growing body of evidence that casein kinases are implicated in GPCR desensitisation.

The ring dove (Streptopeliafi sotia) has proved an excellent animal model for studies in reproductive endocrine-behaviour interactions for many years. It shows bi-parental care and has a stereotyped breeding cycle. Androgen receptor and progesterone receptor immunoreactivity (AR-ir and PR-ir) were localised in the brain of male and female ring doves. Nuclear AR-ir in courting birds was widespread throughout the telencephalong diencephalon, and mesencephalon. Nuclear PR-ir was only localised in discrete regions of the preoptic-hypothalamus of both sexes. In the anterior and posterior hypothalamus, high density of AR-ir and PR-ir neurones was concentrated and co-localised in the nucleus preopticus anterior (POA), nucleus preopticus mediahs (POM), nucleus medialis, pars medianis (POMm), nucleus preopticus paraventricularis magnocellularis (PPM), nucleus hypothalami laterahs posterioris (PLH), and tuberal. hypothalamus (Tu). Investigation of the effects of the breeding cycle on anterior pituitary gland and hypothalamic AR-ir and PR-ir showed a significant (p<0.001) increase in AR-ir cells in courting birds when compared with birds at other stages of the breeding cycle. AR-ir cells in birds brooding young was significantly reduced or absent altogether. In the anterior pituitary gland of both sexes, PR-ir cells increased during courtship and significantly decreased in brooding birds. In the anterior hypothalamus of both sexes, no significant change in PR-ir density was observed. However, a significant increase (p<0.05) of PR-ir neurones was seen 'in the ventral and lateral Tu of incubating male and female doves, respectively, whilst a significant decrease in PR-ir neurones was seen in the ventral region of the Tu in birds brooding young (p<0.001). Although no sex difference was obtained in the number of AR-ir neurones, significantly higher staining intensity for AR-ir was measured in male than female doves, in all hypothalamic regions investigated. Similarly, increased PR-ir staining intensity was measured in courting birds than in birds at other breeding stages. In the anterior pituitary gland and all regions of the hypothalamus the percentage co locahsation between AR-ir and PR-ir changes With breeding cycle. The highest percentage of PR-ir and AR-ir co-localisation (70-909/6w) as obtained in courting birds and the lowest (0.27-24%) in birds brooding young. The association between steroid receptor expression and aromatase P450 enzyme(P450AROM)activity in the brain of the ring dove was also investigated. Sexually experienced ring doves were injected intramuscularly with the aromatase inhibitor fadrozole (0.2 ml of 1 mg n-A-') and paired with saline injected controls in a cage containing a nest bowl and nesting material. The fadrozole/ saline vehicle was administered for 3 days at 12-hour intervals. Saline injected control males displayed aggressive and nest-orientated behaviours whereas fadrozole treated males showed none of these behaviours. Similarly, saline injected control females displayed nest-onented behaviours and were sitting on the nest by day 2, whereas fadrozole injected females showed none of these behaviours. On day 4 of courtship, birds were perfuse-fixed and the brains and anterior pituitary glands subjected to immunocytochernistry for androgen receptor (AR), progesterone receptor (PR) and aromatase (P450ARONýIn* saline treated birds of both sexes, nuclear AR-ir and PR-ir were seen in the PON, PPM, POM, POMm, PLH and Tu. In contrast, fadrozole treatment in both sexes resulted in the complete absence of detectable nuclear AR-ir expression, together with a significant decline in the expression of PR-ir in the POM and POMm, and all regions of the Tu (p< 0.001). A sex difference in PR-ir expression was seen in the POA and PPM (p<0.001) of fadrozole treated birds. Fadrozole administration caused decrease in the percentage co-localisation of AR-ir and PR-ir in both sexes. A high density of AROMir neurones was observed in the POM and POMm of saline injected males, which was found to co-express PR-ir (99%). The viability of maintaining in vitro brain slice preparations was studied with the aim to identify and confirm neural circuits involving the anterior and posterior hypothalamus in the initiation of sexual and parental behaviours in this species. Sections were pressure injected with fluorogold (FG) or Dil (8 -15 nl) in the POA, POM, and the ventral, lateral and dorsal regions of the Tu. Results obtained confirm previous reports of neural connections between the Tu and POA. Neural connections were also seen between the Tu and the nucleus medialis hypothalan-ii posterioris (PMH), and several other regions of the mesencephalon and telencephalon, including the nucleus intercollicularis JCo), nucleus ovoidalis (Ov), dorsolateralis posterior thalami (DLP), archistriatum, pars ventrale (Av) and archistriatum, pars dorsalis (Ad). It is concluded that in the dove, central AR-ir and PR-ir expression and co-localisation are closely associated with the sexual stages of the reproductive cycle. Increases 'in AR-ir neurones in the preoptic-hypothalamus in courting birds are consistent with the behavioural role of androgen acting in this brain area during this time. Similarly, the high PR-ir neurones in the preoptic-hypothalamus are consistent with the progesterone-induced initiation of incubation in this species. The high percentage of AR-ir and PR-ir colocalisation in the preoptic-hypothalamus of courting doves supports previous reports involving progesterone acting in these brain regions to terminate the androgen-dependent aggressive courtship behaviour in male doves. The decrease in the number of co-localised AR-ir and PR-ir neurones M the preoptic-hypothalamus following oestrogen synthesis inhibition suggests a role for oestrogen in the progesterone-dependent termination of aggressive courtship display in males, and models proposing a mechanism involving oestrogen in the orchestration of central systems associated with courtship behaviour in the ring dove are presented. Neural connections between the POA and Tu, and Tu with the PMH, ICo, Ov, DLP, Av and Ad may represent the neuroanatomical pathways involved in the integration of courtship display, vocalisation (nest-coos and/or bow-coos), auditory, propriosceptive signals and gonadotropM secretion in the reproductive cycle of the ring dove. These results are consistent with the hypothesis that the POA, Tu and the midhypothalamus are involved in the orchestration of sexual and parental behaviour of the ring dove.

Fish populations are increasingly under threat by anthropogenic habitat modification. As demands on rivers have increased through increased human activity, resultant watercourse manipulations have altered the natural flow regime. However, it is unclear how diseased fish react to variable flow conditions in terms of their behaviour and swimming ability. This thesis addresses fundamental questions about the interaction between flow hydraulics and fish behaviour using two popular model systems: Trinidadian guppies (Poecilia reticulata) and three-spined sticklebacks (Gasterosteus aculeatus). Firstly it was found that guppies shoaled less when a member was infected with Gyrodactylus turnbulli but the magnitude of this effect was greater in the absence of flow (Chapter 2). Over time, the shoaling behaviour of guppies in the static flow condition reduced as parasite prevalence increased. In the flow condition, however, this effect was not observed, which resulted in higher peak prevalence of the parasite (Chapter 3). Sticklebacks utilised the lower flow velocities near the bed boundary layer to improve anaerobic and aerobic swimming performance but their natural parasite fauna had little effect on their swimming ability (Chapters 4 and 5). Guppies of different size, sex and parasite load utilised different regions around boulders to refuge from undesirable hydraulic conditions (Chapter 6). Finally, the impact of an invasive nematode Anguillicoloides crassus on the swimming behaviour of downstream migrating European eels (Anguilla anguilla) was investigated (Chapter 7). The parasite reduced burst swimming ability of the eels, which may have a knock-on effect for migration. In summary, this thesis demonstrates the importance of flow heterogeneity within a river system to provide shelter for smaller or weaker fish in poor health. River managers need to carefully consider any adaptation of flow regimes to provide appropriate flow conditions for resident species displaying a range of microhabitat requirements.

Microbial biosynthesis of commodity compounds offers a cheaper, greener and more reliable method of production than does chemical synthesis. However, engineering metabolic pathways within a microbe for biosynthesis of a target compound is a complicated process: levels of gene expression, protein stability, enzyme activity, and metabolic flux must be balanced for high productivity without compromising host cell viability. A major rate-limiting step in engineering microbes for optimum biosynthesis of a target compound is DNA assembly, as current methods can be cumbersome and costly. This study aimed to develop a new, synthetic biology tool for rapid DNA assembly that can be applied to engineering and optimizing metabolic pathways for the microbial biosynthesis of commodity compounds. The potential of using serine site-specific recombinases as synthetic biology tools to assemble DNA was investigated and a new DNA assembly method, Serine Integrase Recombinational Assembly (SIRA), using PhiC31 integrase was established. It was demonstrated that SIRA can clone DNA parts ranging in size from 71 bp to 12.7 kb, assemble as many as five DNA parts in a one-pot reaction, facilitate targeted post-assembly modification of an assembled construct and generate variation between DNA constructs in a single assembly reaction. SIRA was used to generate variation between constructs containing genes of the violacein biosynthesis pathway, the lycopene biosynthesis pathway, or the DXP pathway for isoprenoid biosynthesis in E. coli. By studying the phenotypes and genotypes of the constructs generated, it was possible to identify rate-limiting steps within these pathways. Finally, a lycopene-producing in vivo biosensor screen was developed in E. coli to screen DNA assemblies, made with SIRA, encoding genes from the DXP pathway, for enhanced isoprenoid production. By optimizing the expression conditions for assemblies of DXP pathway genes that enhanced isoprenoid production and genes for lycopene biosynthesis in E. coli, 35.78 mg lycopene per gram dry cell weight was obtained - the highest recorded level of lycopene produced from engineering of the DXP pathway alone in E. coli.

X-rays have a long-standing history as an investigative probe in the sciences, and in particular their application to the biological and biomedical sciences has provided an enormous contribution to these fields. Indeed structural biology, the study of the molecules of life at an atomic scale via macromolecular crystallography, has been a major benefactor of advances in x-ray radiation sources. Currently two major bottlenecks exist within this field, the need for well diffracting crystals and radiation damage limitations. The advent of fourth generation x-ray sources, X-ray Free-electron Lasers (XFEL) heralds a shift in the way such experiments are performed. XFELs, due to their high brilliance and ultra short (fs) pulses, hope to decouple radiation dose limitations from spatial resolution by outrunning this radiation damage in short exposures, ‘diffraction before destruction’. This thesis is concerned with exploring experimental methodologies made possible by XFELs, including establishing the experimental infrastructure required at the worlds second XFEL, SACLA, and performing initial experiments. Firstly the potential of performing gas-phase small angle x-ray scattering experiments (gSAXS) is investigated. The current need for gas-phase structural information will be presented and the experimental parameters and projected signal requirements will then be explored. The results of experiments at a synchrotron radiation source with various biomolecules will be presented. It is shown that with the current experimental set-up experiments are fundamentally limited by the signal to noise ratio (SNR) pointing to the necessity of XFEL. Secondly the application of coherent diffractive imaging (CDI) to biological systems at synchrotron and XFEL sources is explored, and the development of experimental systems at both sources is outlined. A method for combining complimentary scattering experiments at both sources is demonstrated and the results of its application to the assembly mechanism of the self-assembling, non-crystalline, macromolecule, the RNAi microsponge, are presented. The microsponge is found to have a nucleating origin leading to a core-shell like nanostructure in the fully formed molecule.

Ageing is defined as an age-related increase in susceptibility to diseases and death and is a complex process that affects every major system at the molecular, cellular and organ level. Although the exact cause of ageing is unknown, there is significant evidence that oxidative stress plays a major role in the ageing process. Skeletal muscle produces oxidants from a variety of different sources with nitric oxide and superoxide being the primary radical species. Nitric oxide is regulated by the activity of nitric oxide synthases, however the sites that modulate changes in superoxide remain unclear. Skeletal muscle ageing is associated with a reduction in muscle mass and strength and leads to a significant vulnerability that opposes healthy ageing. Reports have indicated a positive correlation between tissue concentrations of oxidised macromolecules in skeletal muscle of old individuals, which implies the possible involvement of reactive species in the processes of skeletal muscle ageing. The role of oxidants in skeletal muscle ageing has also been extensively examined in different model organisms, which have undergone genetic manipulations and reports have shown that absence of Cu, Zn superoxide dismutase (SOD1) in homozygotic SOD1 knockout mice, induces an acceleration of skeletal muscle ageing phenotypic changes which further provides support for the implication of radical species in the processes of muscle ageing. The overall aim of the work carried out in this thesis was; i) to develop specific techniques to determine changes in superoxide within the cytosolic and mitochondrial compartment of skeletal muscle, ii) to identify the major sites for superoxide generation in skeletal muscle and iii) to identify the reactive species that are involved in the accelerated loss of muscle mass in the homozygotic SOD1 knockout mouse model and to characterize the changes in redox status and adaptive responses that occur in muscles from the SOD1 knockout mice. The results of carried out in this thesis indicated that the superoxide sensitive fluorescent probes dihydroethidium and MitoSOX Red were capable of selectively detecting changes in superoxide within the cytosolic and mitochondrial matrix of skeletal muscle, respectively. Specific pathway inhibitors and immunolocalisation techniques showed that the major sub-cellular sites contributing to cytosolic superoxide changes in skeletal muscle both at rest and during contractions were the NAD(P)H oxidases. Finally with the use of single isolated muscle fibres from the flexor digitorum brevis muscle, it was concluded that formation of peroxynitrite in muscle fibres was a major effect of lack of SOD1 in SOD1 null mice, which may contribute to fibre loss in this model. Techniques developed in this study to monitor real-time changes in superoxide in the mitochondrial and cytosolic compartment of muscle fibres provided a useful tool to i) examine the sub-cellular pathways that are involved in the regulation of superoxide at rest and during contractile activity in skeletal muscle and to ii) determine the role of superoxide in skeletal muscle degeneration observed in SOD1 knockout mice. These results may have widespread implications for the understanding of diverse scientific areas, including the responses of muscle to exercise training, age-related loss of muscle mass and function, as well as inflammatory or degenerative muscle diseases, such as the muscular dystrophies that are associated with increased levels of oxidative damage.

The mitotic spindle is required to accurately separate the two duplicated copies of the genome between the two daughter cells. Errors that occur during this process can lead to aneuploidy, a hallmark of cancer. Some microtubules of the spindle are bundled by inter-microtubule bridges to form kinetochore fibres (K-fibres), which are responsible for pulling the sister chromatids apart. Previous work has identified one type of inter- microtubule bridge, composed of three proteins: TACC3, ch-TOG and clathrin. These TACC3–ch-TOG–clathrin crosslinkers are important for accurate mitosis and were found to contribute to K-fibre stability. However, these studies used RNAi-mediated depletion to study the functions of these proteins at metaphase. RNAi-mediated depletion is slow, and it is therefore difficult to separate the roles of the proteins at early mitosis versus metaphase. For instance, the depletion phenotype at metaphase may have been due to the absence of the protein at metaphase, or to the assembly of a defective mitotic spindle in early mitosis. Here, knocksideways (KS) was used to deplete TACC3 in under 5 minutes from fully assembled spindle at metaphase, and trap the protein on the external membrane of mitochondria. TACC3 KS also caused the rerouting of both ch-TOG and clathrin, without affecting other spindle proteins. This depletion was supported by a concomitant reduction of inter-microtubule bridge frequency in K-fibres. When TACC3 KS was performed at early mitosis, cells displayed severe delays in aligning chromosomes. When TACC3 KS was performed at metaphase following a normal prometaphase, anaphase onset was delayed. In these cells, Mad2 on a subset of kinetochores indicated that the spindle checkpoint was active, and K-fibre tension was reduced. However, both a cold-stable K-fibre assay and electron microscopy analysis of K-fibres indicated that there was no significant loss of K-fibre microtubules, even after prolonged removal at metaphase. Furthermore, TACC3 KS at metaphase significantly altered kinetochore microdynamics and reduced spindle length. These results indicate that TACC3–ch-TOG–clathrin crosslinkers are important for mitotic spindle assembly, but not for K-fibre stability at metaphase. However, the complex is required for K-fibre function at metaphase, satisfaction of the spindle checkpoint, most likely by maintenance of K-fibre tension.