Accurate and robust monitoring of product and reactants in a complex bioconversion stream is essential for the development of effective process control strategies. To monitor a microbially-catalysed Baeyer-Villiger bioconversion of a cyclic ketone to an optically pure lactone, a near infrared (NIR) spectroscopic method has been developed. The reaction, catalysed by cyclohexanone monooxygenase from Acinetobacter calcoaceticus (expressed in Escherichia coli) is characterised by substrate (ketone) and product (lactone) inhibition at relatively low concentrations. Quantitative multivariate calibration of a NIR spectrophotometer for ketone and lactone resulted in a standard error of prediction (SEP) at-line of 0.088 and 0.110 g/l-1 and on-line of 0.130 and 0.180 g/l-1 , respectively. The directed modification of quantitative models, by the inclusion of spiked process samples improved the SEP for lactone prediction where bioprocess development meant existing NIR models were not relevant. The monitoring and control of the Baeyer-Villiger bioconversion by NIR has allowed intermittent feeding of ketone such that the concentration of substrate does not rise above 0.6 g/l-1. Using this feeding strategy 5.7 g/l-1 of lactone product has been produced. This represents a 2-fold increase in productivity. The application of a technique to monitor analytes at low concentration demonstrates the utility of NIR for control of biotransformation processes.

The defensins have been shown to be an important component of the innate immune system across many species from plants to man. To date a total of 14 beta defensins have been identified within the chicken genome and anti-microbial activity of an array of these peptides against pathogens has been demonstrated. The innate immune system has been shown to be important in chickens during the first week of life when exposure to pathogens in the environment occurs and the adaptive immune system is not fully developed. The research presented in this thesis attempts to investigate the effects of bird age (aged 0, 7, 14 and 35 days), genetics and rearing environment on components of the innate immune system. A farm trial was performed using three lines of Aviagen birds (lines X, Y and Z) and two distinct rearing environments (low and high hygiene farms). To determine which of the 14 avian beta defensins (AvBD) to investigate, a panel of potential single nucleotide polymorphisms (SNPs) within the AvBD locus was submitted as part of an Aviagen Ltd genomics initiative. Frequencies of the polymorphisms across the three lines of birds were determined. From these data, gene expression of AvBDs1, 4 and 10 were fully investigated using end-point PCR and then quantitative real-time PCR (qRT-PCR). A pronounced finding from the qRT-PCR was the marked intra and inter-group variation in gene expression levels, which lead to few statistical significant differences. All three genes were shown to be expressed across a panel of ten tissues analysed, but distinct patterns were also seen. Significantly AvBD1 gene expression in the duodenum indicated that of the 7 day old birds the line X birds reared in the low hygiene environment had the highest level of gene expression. In relation to AvBD4 gene expression, the highest level was observed in the spleen of the 0 day old birds, but overall environment did not appear to affect AvBD4 gene expression of the tissues examined. High levels of AvBD10 gene expression were observed in bird kidney and testicle tissue, but again environment in the case of the former tissue did not appear to statistically affect gene expression levels, the YH 7 day old bird testicle samples did have statistically significant higher expression levels compared to the other groups. The SNP analysis revealed three non-synonymous polymorphisms within the AvBD1 mature peptide locus. The three lines of birds had quite different patterns of these polymorphisms and so three different forms of the peptide along with a single form of the AvBD10 peptide were synthesised using a bacterial hyper-expression system. Peptide levels were quantified using an ELISA and subsequently tested in a bacterial time-kill assay using Salmonella enterica serovar Typhimurium phoP, Staphylococcus aureus and two strains of Enterococcus faecalis. All recombinant peptides showed anti-microbial activity against the bacteria tested. The exception was a clinical isolate of E. faecalis which showed resistance to the killing activities of recombinant AvBD10 peptide. Duodenal gut protein extracts were also tested using the same bacterial assay and marked differences in the anti-microbial activities of these samples were seen. The samples taken from the day 0 birds were found to have significant anti-microbial activity compared to those of the older birds. LC/MS identified differences in the proteomes of the respective gut extracts. These data support that bird genetics, age and the environment have an effect on AvBD gene expression and gut anti-microbial activity. These differences are not uniform for all genes and groups of birds, but clear patterns were observed.

Grid cells fire in multiple locations arranged in a hexagonal symmetry and may provide the ‘spatial metric’ for the hippocampal cognitive map. It is unclear how their grid-like firing patterns are generated, and whether they represent one end of a wider continuum of locational firing patterns or an entirely distinct unique pattern. In this thesis I report a range of spatially periodic firing patterns including canonical grid cells in neuronal populations recorded from the medial entorhinal cortex and adjacent parasubiculum of rats foraging for food in simple enclosures. Two-dimensional spatial Fourier analysis revealed that the firing patterns of many cells of this larger population were composed of a small number of periodic bands. Configurations of periodic bands with a 60º organisation corresponded to grid cells and had the most stable firing properties. Other spatially periodic patterns showed a higher degree of variability in the orientations of their spatial components and significantly diverged from hexagonal symmetry. In general all the components of spatially periodic cells tend to align within each animal regardless of their symmetrical properties. Firing patterns in a single cell occasionally changed between hexagonal and non-hexagonal spatially periodic patterns, pointing to a single mechanism for the formation of the diverse patterns. My results describe a general class of spatially periodic cells in the parahippocampal formation, including grid cells as the most regular and stable, and suggest that they are formed from a restricted set of elemental periodic band-like components.

Animal cells endure dramatic actin-dependent changes in shape as they progress through mitosis – they round up at mitotic entry, elongate at anaphase and split into two at cytokinesis. In this thesis I explore the role of Moesin, an actin-membrane crosslinker and the sole ERM protein expressed in Drosophila, in orchestrating rearrangements of the actin cortex and morphological changes in epithelial cells undergoing mitosis. To perform my studies I used the fly notum and sensory organ precursor (SOP) cells therein as a model system. In this thesis I show that Moesin is required for the stabilisation of the actomyosin cortex at metaphase. This mechanism is dependent upon phosphorylation of Moesin by the Slik kinase, which activates the ERM protein. Reduced levels of Moesin or Slik lead to myosin-II-driven cortical instabilities. Cortical stabilisation in mitotic SOP cells ensures the efficient accumulation of fate determinants at the plasma membrane. At mitotic exit, a pool of active, phosphorylated Moesin is lost from the cell poles, thereby triggering polar relaxation and initiating anaphase cell elongation. These two events precede furrow formation, are independent of centrosome or astral microtubules-derived signals, and are induced by proximity of the segregating chromosomes to the cell poles. I show that a pool of kinetochore-localised PP1-87B phosphatase and its regulatory subunit Sds22 inactivate cortical Moesin and elicit the dismantling of the actomyosin cortex at mid-anaphase. Cells with reduced amounts of PP1-87B or Sds22 fail to clear Moesin and actin from the anaphase poles. Importantly, these defects in polar relaxation are mimicked by the expression of a constitutively active form of Moesin in fly tissues. Finally, I demonstrate that delocalisation of PP1/Sds22 from the kinetochores via KNL1 depletion abolishes polar blebbing at anaphase and impairs cell elongation. My work shows how the dynamic regulation of Moesin activation and localisation controls shape changes in cells undergoing mitosis. Moreover, it sheds light on a novel mechanism of polar relaxation at anaphase, in which a kinetochore-derived signal instructs the cell cortex to become polarised, thereby initiating cytokinesis.

Wnt signalling is central to many early developmental processes including embryonic patterning, programmed cell death and cell migration. Recent studies have implicated Wnt signalling in the formation of neuronal connections in the nervous system. Wntl a regulates synapse formation in the cerebellum by inducing presynaptic differentiation characterized by axonal remodelling and presynaptic assembly. A very similar gene, Wnt7b regulates dendritic development in hippocampal neurons, but can also trigger axonal remodelling. These different responses are due to the activation of different signalling pathways. We aim to identify the receptors triggering these processes in neurons. Intracellular signalling by Wnts is initiated by the activation of their seven transmembrane receptors Frizzled (Fz). Three known Wnt signalling pathways may be activated: the canonical, the planar cell polarity and Calcium pathways. Ten Fz receptors and 19 Wnts have been identified in the mouse genome. To begin to address what Fz receptors are used by Wnts, we examined the pattern of expression of Wnt 7a and Wnt 7b together with several Fz receptors during postnatal brain development. We found that Wnt7a,fz7, and fz3 are expressed in the postnatal and adult cerebellum. Wnt7b and fz3 are highly expressed in the postnatal hippocampus. These overlapping patterns of expression led us to investigate the ability of Wnt7a and Wnt7b to bind to the cell surface of HEK293 cells expressing the ligand-binding domain of Fz receptors. Binding of Wnt7a an -7b to Fz-3, -5, -7 and -8 was tested. Wnt7b binds Fz3 and Fz5, whilst Wnt7a binds Fz7 and Fz3. Signalling activity was then assessed by measuring TCF/LEF mediated transcription (Top-Flash assay) and by the increased levels of 6-catenin. Wnt-7a is able to activate the canonical pathway in Fz7 and Fz3/LRP6 transfected HEK293 cells. These studies highlight the idea that activation of the canonical / p-catenin pathway by Wnt7a can be mediated by Fz7 and the LRP6/Fz3 complex.

Alterations in RhoA (RHO-1) signalling cause neuronal signalling defects in both mammals and C. elegans. In the case of C. elegans, exaggerated RHO-1 signalling in cholinergic neurons (nRHO-1*) has numerous effects that include increased release of acetylcholine (ACh) and exaggerated body curvature during locomotion. A suppressor screen identified the mutation, nz99, that suppresses nRHO-1* exaggerated curvature, but not increased ACh release. Non-neuronal defects caused by heat-shock, expression of RHO-1* (sterility, death, tail-swelling and protruding vulva) were not suppressed, suggesting that DAT-1 is required only for RHO-1 neuronal signaling. Whole genome sequencing and rescue experiments identified the nz99 suppressor as a mutation in the dopamine transporter DAT-1. DAT-1 is a negative regulator of dopamine (DA) in both mammals and C. elegans by transporting extracellular DA into cells. dat-1 mutations were unable to suppress nRHO-1* locomotion defects when DA synthesis was inhibited either pharmacologically, using the drug reserpine, or genetically by a cat-2, (tyrosine hydroxylase) mutation. This is consistent with a model in which elevated DA signaling acts to inhibit the locomotion defects of nRHO-1*. Exogenous DA causes a dose dependent slowing of locomotion in C. elegans, and this was suppressed by nRHO-1*. Thus nRHO-1* and DA co-suppress each other's locomotion behaviours, suggesting that DA and RHO-1 signalling act in parallel to control locomotion. C. elegans possess at least four DA receptors of the G protein coupled class (DOP-1-4) that fall into either the D1 (DOP-1, 4) or the D2 class (DOP-2, 3). All four receptors are required for elevated DA signaling to suppress nRHO-1* locomotion defects. In other studies, the D1 and D2 classes of DA receptor have been shown to act antagonistically in controlling locomotive behaviours including, swimming, basal slowing and paralysis. My data suggests that a co-ordinated DA response that is mediated by multiple DA receptors, possibly acting in numerous cell types, also acts to modulate locomotion in C. elegans possibly by altering both small synaptic vesicle and dense core vesicle release.

Cells embedded within tissues respond to mechanical, chemical and biological signals. However, the detail of how mechanical forces are transmitted to cells is poorly understood at present and represents a key missing link in Tissue Engineering. As cells attach to the fibrils in fibroblast-seeded 3D collagen scaffolds they generate contractile forces to levels, which depend on cell type, attachment, density, growth factors and matrix stiffness. The aim of this study was to use external applied strain to increase matrix stiffness in collagen constructs. Embedded resident cells (from three different sites) were then subjected to specific mechanical loading regimes in scaffolds of increasing stiffness and matrix remodelling genes quantified as markers of mechanoregulatory cellular response. Mechanical responses of cells were also quantified as contraction profiles over time. Our findings indicated that collagen got stiffer with application of high strains and visco-elastic properties resulted in minimal transfer of applied loads as recorded by movement of indwelling markers. The mechanical and molecular responses of three different cell lineages: human dermal (HDF), neonatal foreskin fibroblasts (HNFF) and human bone marrow stem (hBMSC) cells seeded in constructs of increased stiffness was tested. Results indicated that in HNFFs contraction was predominantly attachment-dependent while in HDFs it was predominantly stiffness-dependent. hBMSCs showed differential response to serum levels. Molecular responses in progressively stiffer constructs investigated were MMP-2, MMP-3, MMP-9, TIMP- 2,COL-l,COL-3 and IGF-1. Different cell types expressed specific variations in gene regulation. The effect of specific mechanical loading (slow and fast ramp) regimes on regulation of matrix remodelling genes also showed a lineage dependent response. The major impact of this project has been the identification of a strong co-relation between substrate stiffness, mechanical loading and regulation of key ECM turnover genes. This knowledge is crucial to successful tissue engineering outcomes. The differential lineage dependent response is a key finding and will have to be tailored depending on cell source and specific outcomes desired. regimes on regulation of matrix remodelling genes also showed a lineage dependent response. The major impact of this project has been the identification of a strong co-relation between substrate stiffness, mechanical loading and regulation of key ECM turnover genes. This knowledge is crucial to successful tissue engineering outcomes. The differential lineage dependent response is a key finding and will have to be tailored depending on cell source and specific outcomes desired.

The epidermal growth factor receptor (EGFR) is best known for its role in triggering a variety of intracellular signalling pathways that promote cell growth, proliferation, differentiation and migration following the binding of its ligand (EGF). As a result of this, over-activation of EGFR can contribute to carcinogenesis. In parallel with receptor activation, ligand binding initiates receptor internalisation, which provides an important mechanism for negatively regulating the duration and potency of signalling by kick-starting EGFR traffic to the lysosome where it is degraded. However, a small proportion of EGFR can be recycled back to the plasma membrane, prolonging signalling. Another controversial fate has been suggested for the EGFR: traffic to the nucleus where it is believed to promote cell cycle progression, proliferation and DNA repair. In addition to ligand-dependent activation and internalisation of EGFR, these processes can also occur in a ligand-independent manner, following various stresses, including chemotherapy, radiotherapy and ultraviolet light C (UVC). The fate of EGFR following ligand-independent stimulation has not been fully characterised, but in some instances it has been reported to traffic to the nucleus. This would have therapeutic implications, as DNA damage following chemotherapy or radiotherapy could be repaired, resulting in the development of resistance to therapy. In this thesis I aimed to establish tools that inhibited stress-induced EGFR endocytosis in order to test the hypothesis that the fate of stress-induced EGFR regulates EGFR signalling, DNA repair and cell survival. I focused on UVC-induced internalisation, as this occurs rapidly and synchronously and shares a mechanism of internalisation with the chemotherapeutic drug cisplatin. I have shown that following UVC irradiation or cisplatin treatment of HeLa cells, EGFR is internalised and retained in a peri-nuclear compartment without being degraded or translocated to the nucleus. This internalisation depends on the activity of both p38 and Src but not the EGFR tyrosine kinase. Electron microscopy revealed EGFR to be sequestered in multivesicular endosomes/bodies (MVBs). Although these MVBs were morphologically indistinguishable from those that traffic ligand-stimulated EGFR to the lysosome, stress exposed EGFR showed little degradation despite apparent near normal lysosomal function. Stress-induced EGFR endocytosis was not directly linked to DNA damage as ionizing radiation and UVA treatment, which induced extensive DNA damage but did not activate p38, did not induce detectable EGFR internalisation in HeLa cells. EGFR internalisation also correlated with subsequent cell death, as only doses of UVC that induced cell death also induced EGFR endocytosis. Stress-induced EGFR internalisation was abrogated upon depletion of the clathrin adaptor complex AP-2, or mutation of the two AP-2 interaction motifs in the cytoplasmic domain of the EGFR, indicating that this endocytosis occurs via clathrin-coated pits. Inhibition of stress-induced EGFR endocytosis demonstrated that endocytosis of EGFR was necessary to maintain EGFR tyrosine kinase activity, downstream signalling and delay entry into apoptosis. In conclusion we have uncovered a potential role for EGFR in the development of therapeutic resistance. By understanding the molecular mechanisms affecting the endocytic fate of EGFR following stress exposure, it may be possible to design strategies that divert patients from such resistance in the future.

Cellular senescence, the irreversible loss of proliferating capacity of somatic cells, is an important tumour suppressor mechanism but also driver of ageing. These somehow contradictory functions are dependent on the development of the so-called senescent phenotype, which involves over-production of pro-inflammatory and pro-oxidant signals, however the exact mechanisms underlying its induction remain incompletely understood. In this thesis we aimed to understand how mitochondria and pro-inflammatory factors interact during senescence and how they contribute to the senescent phenotype. Firstly, we show that mitochondria are critical for the establishment and maintenance of cell senescence. Elimination of mitochondria rejuvenated senescent human fibroblasts, abrogating the pro-inflammatory phenotype, heterochromatin foci and expression of cyclin-dependent kinase inhibitors p21 and p16. Importantly, a considerable percentage of these cells were able to resume proliferation. Mechanistically, we show that mTORC1 integrates signals from the DNA damage response towards PGC-1β-dependent mitochondrial biogenesis, playing a causal role in the development of senescence. Secondly we show that inhibition of IL-8, a prominent proinflammatory cytokine of the SASP, partially abrogated the senescent phenotype by reducing mTOR-dependent mitochondrial mass and ROS production during senescence. Finally, we demonstrate that inhibition of mitochondrial content in vivo by either rapamycin or PGC-1β deletion prevents age-dependent increase in senescence in mouse liver. Our results suggest mitochondria as an important target for interventions aiming to reduce the load of senescent cells in ageing tissues.

Nitric oxide (NO) is constitutively generated by cardiac myocytes and has important roles in cardiac function, including modifying L-type Ca2+ currents (ICa,L). The precise nature of this modification remains elusive with NO reported to increase, reduce or have biphasic effects on ICa,L. Here I explored the effects of NO signalling on ICa,L in both active period and resting period myocytes recorded from guinea pig ventricular myocytes using the perforated whole-cell switched voltage-clamp technique to maintain intracellular signalling pathways. Both cGMP-dependent and S-nitrosylation pathways were investigated. Isoprenaline (100 nM) significantly increased peak ICa,L by about two fold. Subsequent application of NO, using the NO donor SNAP, significantly decreased this enhanced ICa,L but had little effect on basal ICa,L. In contrast to these results obtained from active period myocytes, NO did not inhibit isoprenaline enhanced ICa,L in resting period myocytes. In active period myocytes NO inhibition of isoprenaline enhanced ICa,L was maintained in the presence of ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one), a soluble guanylyl cyclase (sGC) inhibitor. Direct activation of sGC or peripheral guanylyl cyclase independently of NO by BAY 60-2770 or ANP respectively, however, gave results similar to those observed with NO; although in some cells, BAY 60-2770 did not reduce isoprenaline enhanced ICa,L. Thus direct activation of sGC mimics the effect of NO, yet inhibiting sGC did not abolish NO reduction of isoprenaline enhanced ICa,L. These results suggest that NO modulates ICa,L through more than one mechanism. To investigate the S-nitrosylation pathway, denitrosylation was inhibited using N6022, a blocker of S-nitrosoglutathione reductase, an enzyme involved in denitrosylation. This treatment, either completely abolished or slowed significantly the rate of development of isoprenaline enhancement of ICa,L in cells exposed previously to NO. In conclusion, NO inhibition of isoprenaline enhanced ICa,L involves at least two signalling pathways; a cGMP-dependent and the S-nitrosylation pathway in active period myocytes.