Many high throughput sequencing protocols for RNA and DNA require that the polynucleic acid is fragmented so that the identity of a limited number of nucleic acids of one or both of the ends of the fragments can be determined by sequencing. The nucleic acid sequence allows the fragment to be located within the genome, and the fragment distribution can then be used for a variety of different purposes. In the case of DNA this includes identifying the locations where specific proteins are bound to the genome. In the case of RNA this includes quantifying the expression levels of different gene variants or transcripts. If the locations of the polynucleic acid fragments are partly determined by the underlying nucleic acid sequence this could bias any results derived from the data. Unfortunately, such sequence dependencies have already been observed in the distribution of both RNA and DNA fragments. Previous analyses of such data in order to reduce the bias have examined the role of regional characteristics such as GC bias, or the bias towards a specific sequence at the start of the fragments. This thesis introduces a new method for modelling the bias which considers the degree to which the nucleotide sequence affects the likelihood of a fragment originating at that location. This shows that there is often not a single bias characteristic, but multiple, alternative sequence biases that coexist within a single dataset. This also shows that the nucleotide sequence immediately proximal to the fragment also has a significant effect on the fragment likelihood. This new approach highlights characteristics that were previously hidden and provides a more powerful basis for correcting such bias. Multiple alternative sequence biases are observed when both RNA and DNA are fragmented, but the more detailed information provided by the new technique shows in detail how the characteristics are different for RNA and DNA and indicates that very different molecular mechanisms are responsible for the biases in the two processes. This thesis also shows how removing the effect of this bias in ChIP-seq experiments can reveal more subtle features of the distribution of the fragments. This can provide information on the nature of the binding between proteins and the DNA with per-nucleotide precision, revealed through the change in likelihood of the DNA fragmenting at each position in the binding site. It is also shown how the model fitting technique developed to analyse sequence bias can also be used to obtain additional information from the results of ChIP-chip experiments. The approach is used to find the nucleotide sequence preference of DNA binding proteins, and also the cooperative effects associated with binding at multiple binding sites in close proximity.

The protein DC-SIGNR (Dendritic-cell specific ICAM3 grabbing non-integrin related) is a C-type (calcium-dependent) lectin, which binds highly-branched mannose oligosaccharides. DC-SIGNR interacts with a range of deadly diseases via surface glycans on pathogenic glycoproteins, and the ability of DC-SIGNR to increase the rate of infection of viruses including human immunodeficiency virus (HIV) and hepatitis C virus (HCV) makes the study of DC-SIGNR/oligosaccharide interactions very attractive. The research described in this thesis sought to gain insight into the calcium and ligand binding properties of the DC-SIGNR carbohydrate recognition domain (CRD) in solution by utilising solution state nuclear magnetic resonance spectroscopy (NMR). A protocol for the production of uniformly 15N /13C labelled DC-SIGNR CRD was developed, allowing the acquisition of heteronuclear NMR experiments and the first assignment of the calcium-bound (holo) DC-SIGNR CRD to be reported. The assignment has allowed investigation of calcium and glycan binding, as well as the pH dependence of the DC-SIGNR CRD. The data presented in this thesis reveal that the DC-SIGNR CRD is highly dynamic in the calcium-free state, with the addition of calcium resulting in global conformational and dynamic changes throughout the CRD. While calcium binding hinders the protein dynamics (particularly in the calcium binding regions), a large degree of mobility remains. The evidence that ligands are released at low pH suggests that DC-SIGNR may act as an endocytic receptor. In addition to calcium binding, interactions of the DC-SIGNR CRD with a range of ligands were investigated. In particular, interactions with the oligosaccharide Man9GlcNAc (present on the HIV viral envelope) are described, representing the first direct study of the CRD interacting with a diseaseassociated ligand. The glycans employed in this study all bind to the primary calcium binding site, supporting previous crystal data. However, each glycan displays distinct patterns of chemical shift perturbations implying that they each have different, extended binding modes. Particularly striking is the difference between the disease-associated Man9GlcNAc ligand and the ligand present in a previously published crystal structure, (GlcNAc)2Man3. An investigation of the dynamics of the CRD in the holo form and bound to the ligand Man5 shows that the CRD is highly dynamic and that glycan binding further hinders, but does not abolish, the molecular motions. The dynamics data also suggests that a ligand-induced conformational change may occur and indicates potential new binding sites which are not present in any published crystal structures. The dynamic nature of the DC-SIGNR CRD may explain the wide range of ligand specificities and affinities of the C-type lectin scaffold and suggests that the study of the ligand binding properties and dynamics of proteins such as DC-SIGNR in solution is essential to further understanding of this class of proteins.

When we think about neural cells, we immediately recall the wealth of electrical behaviour which, eventually, brings about consciousness. Hidden deep in the frequencies and timings of action potentials, in subthreshold oscillations, and in the cooperation of tens of billions of neurons, are synchronicities and emergent behaviours that result in high-level, system-wide properties such as thought and cognition. However, neurons are even more remarkable for their elaborate morphologies, unique among biological cells. The principal, and most striking, component of neuronal morphologies is the dendritic tree. Despite comprising the vast majority of the surface area and volume of a neuron, dendrites are often neglected in many neuron models, due to their sheer complexity. The vast array of dendritic geometries, combined with heterogeneous properties of the cell membrane, continue to challenge scientists in predicting neuronal input-output relationships, even in the case of subthreshold dendritic currents. In this thesis, we will explore the properties of neuronal dendritic trees, and how they alter and integrate the electrical signals that diffuse along them. After an introduction to neural cell biology and membrane biophysics, we will review Abbott's dendritic path integral in detail, and derive the theoretical convergence of its infinite sum solution. On certain symmetric structures, closed-form solutions will be found; for arbitrary geometries, we will propose algorithms using various heuristics for constructing the solution, and assess their computational convergences on real neuronal morphologies. We will demonstrate how generating terms for the path integral solution in an order that optimises convergence is non-trivial, and how a computationally-significant number of terms is required for reasonable accuracy. We will, however, derive a highly-efficient and accurate algorithm for application to discretised dendritic trees. Finally, a modular method for constructing a solution in the Laplace domain will be developed.

The actions of many neuromodulators induce changes in synaptic transmission and membrane excitability, and many of these effects are well documented in neurons across the CNS. Adenosine acts as a powerful modulator across the CNS and while its actions have been characterised in some neurons in the neocortex, its effects on excitatory transmission in layer 5 remain unstudied. Adenosine has been implicated in the modulation of spontaneous activity generated in the layer 5 excitatory network, thus understanding its actions in this area are of substantial importance. This study used a combined approach of paired intracellular recordings and quantitative modelling to investigate the actions of adenosine on thick-tufted layer 5 pyramidal neurons in the rat somatosensory cortex. Adenosine was found to powerfully suppress synaptic transmission between these neurons and the changes in synaptic dynamics could be precisely captured as a change only in probability of release in a simple phenomenological model. Recordings conducted at three post-natal ages provide evidence that an increased tone of endogenous adenosine is responsible for the previously described developmental shift in short-term dynamics and reliability of this synapse. The data illustrates both that this endogenous activation of A1 receptors is highly heterogeneous, with variation between neighbouring synapses, and that it plays a significant role in EPSP parameters observed at mature connections. An investigation into adenosine's post-synaptic actions using an approach that measures the neurons' I-V response to naturalistic current inputs demonstrates how adenosine's actions on membrane excitability translate to a strong suppression of spiking. Simultaneous dendritic and somatic recordings demonstrate that this effect is enhanced when current is injected from the dendrite and that back-propagating bursts of action potentials are selectively suppressed by adenosine. As a whole the work illustrates that the effects of adenosine can be well captured by mathematically tractable quantitative models.

Tauopathies are a group of neurodegenerative diseases associated with the build-up of intracellular aggregates of the microtubule-associated protein (MAP) tau, called neurofibrillary tangles (NFT). The tau found in NFTs is known to be highly phosphorylated, and as such a number of tau kinases have been examined for their ability to affect tau toxicity in animal models, including members of the AMPK-related kinase family. Two members of this family, the brain-selective kinases (BRSKs), have been shown to phosphorylate tau in vitro and in vivo in an LKB1 and CaMKKα-dependent manner. BRSKs are prevalent in the central nervous system, in which they modulate neuronal polarisation via tau. They have also been shown to dramatically increase 0N4R tau toxicity in a Drosophila model. In order to determine the importance of phosphorylation to tau toxicity in this model, I have generated a group of 2N4R tau phosphorylation mutants. Based upon in vitro data linking phosphorylation at T212 to tau aggregation, and S262 to microtubule dissociation, these two residues underwent both non-phosphorylatable and phosphomimetic mutation and were inserted into Drosophila. Each of these mutations was found to alter tau toxicity upon expression in the fly eye, as measured by disruption of ommatidia, with the T212D and S262A mutations producing a milder phenotype. Each of these sites on tau has previously been shown to be a target of phosphorylation by BRSK2 in vitro. Through generation of a T212D S262D double mutant, I was able to investigate whether phenotypic changes caused by BRSK2 were due to phosphorylation. BRSK2 was found to have a different effect on toxicity caused by 2N4R tau, causing a reduction in toxicity. This was found to be comparable to the phenotype of the double mutant. However, examination of the ability of BRSK2 to modulate the toxicity of the single tau mutants demonstrated a more complex phosphorylation profile, also dependent on other residues. Further experiments examined the consequences of tau and BRSK2 expression in the CNS. This demonstrated the toxicity of BRSK2 overexpression, in the absence of tau, in these tissues. Given that BRSK2 overexpression alone in the eye led to no observable phenotype, this tissue-dependent toxicity of the kinase demonstrates the importance of the choice of model in such studies. In addition, a deficiency screen for modulators of 0N4R tau/BRSK2 toxicity in the eye has generated multiple leads, including the fly homologue of the actin-binding protein profilin. This project has demonstrated the inequivalence of several phosphorylation sites in vivo, as well as an isoform-dependent aspect of tau toxicity. These data suggest that the influence of tau phosphorylation on toxicity is complex; expansion of the work presented here could help decipher the code of tau phosphorylation.

The family of “stress” peptides that includes CRH and UCNs are emerging as important regulators of the homeostatic mechanisms regulating energy balance and metabolism. These peptides exert well documented central anorectic and thermogenic actions in controlling food uptake and optimise energy losses. Furthermore, CRH acting through specific G-protein coupled receptors, CRH-R1 and R2 can target multiple peripheral tissues such as skeletal muscle and adipose tissue to influence important metabolic pathways. Two types of adipose tissue exist in mammals: WAT and BAT. Since WAT is the largest energy reserve in mammals and BAT can utilize energy through adaptive thermogenesis, one of the goals in this study was to identify the presence of CRH system components in adipose tissue. Real time RT-PCR and immunofluorescence demonstrated that CRH-Rs as well as CRH, UCN-I, and UCN-II are expressed in both WAT and BAT, raising the possibility that CRH and UCNs are important regulators of energy storage and adaptive thermogenesis. Also the functional roles of CRH-Rs in adipose tissue were investigated. Using an experimental paradigm the T37i fibroblast that can differentiate into brown adipocyte, it was demonstrated that CRH at low (nanomolar) but not high (submicromolar) concentrations stimulated a signaling pathway involving the AC/cAMP/PKA/AMPK signaling cascade that regulates downstream phosphorylation of HSL. This was associated with a significant translocation of HSL toward lipid droplets and association with perilipin, as demonstrated with immunofluorescence. Studies applying quantitative RT-PCR also suggested that CRH-R1 appears to regulate genes important for adaptive thermogenesis, whereas CRH-R2 likely regulates brown adipocyte formation. Further analysis using an experimental paradigm the 3T3L1 fibroblast that can differentiate into white adipocyte showed that exposure of 3T3L1 cells to UCN-II (a specific CRH-R2 agonist) or NBI-27914 (a CRH-R1 specific antagonist) were able to induce morphological and biochemical characteristics suggesting adipocyte differentiation to a “beige” phenotype in white preadipocytes/adipocytes. Thus, CRH-R1 and R2 could be of potential importance in maintenance of energy homeostasis. Moreover, in vivo analysis showed that CRH system seems to demonstrate a certain degree of plasticity in response to stress perturbation. For instance, HFD significantly repressed the expression of CRH-Rs and their agonists, whereas food deprivation dramatically increased their expression. The analysis of quantitative RTPCR demonstrated that this activation of CRH system might be associated with induction of ‘beige’ cells in white fat depots. Since CRH-R1 KO mice displayed a lean phenotype and resistance to HFD-induced fat accumulation and these phenotypes can be reversed by supplementation of corticosterone, role of CRH-R2 in adipose tissue of these KO mice was investigated. Data showed that CRH-R2 activation likely induced BAT activity and transdifferentiation from WAT to BAT in CRH-R1 KO mice. Corticosterone reversed these changes in KO mice via potential suppression of CRH-R2.

Adenosine receptors (ARs) modulate many cellular and systems-level processes in the mammalian CNS. However, little is known about the trafficking of ARs in neurons, despite their importance in controlling seizure activity and in neuroprotection in cerebral ischemia. To address this I examined the agonist-dependent internalisation of C-terminal GFP-tagged A1R, A2AR and A3R in primary hippocampal neurons and compared findings to CHO cells. Furthermore, I developed a novel super-ecliptic pHluorin (SEP)-tagged A1R which, via the N-terminal SEP tag, reports the cell-surface expression and trafficking of A1R in real-time. I demonstrate the differential trafficking of ARs in neurons: the A3R internalised rapidly, with the A1R internalising more slowly, and with little evidence of appreciable A2AR trafficking over the time-course of the experiments. These findings were consistent with trafficking data in CHO cells and previous literature. Futhermore, the novel SEP-A1R construct revealed the time-course of internalisation and recovery of cell surface expression to occur within minutes of agonist exposure and removal, respectively. These observations reveal the labile nature of cell surface expression of neuronal adenosine A1R and A3Rs. Given the high levels of adenosine in the brain during seizures, internalisation of the inhibitory A1R may result in hyperexcitability, increased brain damage and the development of chronic epileptic states. To test this I monitored trafficking of A1R in response to two seizure conditions, kainic acid and Mg2+free-/high K+. I observed following acute Mg2+free-/high K+ treatment an increase in dendritic puncta consistent with A1R internalisation. In addition to monitoring changes to GFP-tagged AR constructs I attempted to elucidate the effect of agonist exposure on native A1R. Electrophysical recordings revealed that localisation of ARs may impact on receptor regulation, since no evidence of internalisation was observed. This suggested that post-synaptic A1R may be more resistant to internalisation as previously noted in the literature. This study provides an initial study into the regulation of adenosine receptors in hippocampal neurons and has developed tools that will provide useful in further studies to elucidate the regulation of ARs during pathological conditions.

Background: Salt reduction and universal salt iodisation programmes are implemented worldwide to prevent cardiovascular disease and iodine deficiency disorders, respectively. Concerns have been raised regarding the potential policy conflicts, and a programme coalition is proposed by the World Health Organization to optimise salt and iodine intakes at population level. This study aimed 1) to estimate population salt intake and iodine status in index countries; 2) to investigate the association between salt and iodine intakes; 3) to assess the impact of salt intake modification on iodine status; 4) to estimate the determinants of and potential geographical variation in salt and iodine intakes where data are available; and 5) to provide suggestions to policy makers. Data and Methods: In the ecological analysis, national estimations of salt and iodine intakes were extracted from international organisation databases and published papers. Three case studies used population level data obtained from the Kumasi Salt Reduction Study in Ghana, the Third United States National Health and Nutrition Examination Survey (NHANES III) and the 2000-01 UK National Diet and Nutrition Survey 19-64 years (NDNS). Linear regression was used in the Kumasi analysis, and Bayesian geo-additive models were used in the other two analyses by accounting for the spatial effect and important linear and nonlinear risk factors. Results: Salt intake varied between countries, with Kumasi lower than the western countries. Iodine status also varied by country, but with no consistent association with salt intake. A moderate salt reduction programme is unlikely to have a major impact on iodine status in countries committed to universal salt iodisation, provided that iodine concentration is titrated to actual salt intake, maximum coverage is achieved as in China and iodised salt becomes part of food processing. At least in Britain, high salt intake is associated with low socioeconomic status, irrespective of geographic location. Conclusions: Policy-makers may therefore need to adjust iodine content in salt in accordance with each country’s context. The Bayesian geo-additive models are useful for monitoring and evaluating salt reduction and iodine supplementation.

Protein kinase C δ (PKC-δ) is a novel member of the PKC family of serine-threonine kinases. PKC-δ structure is widely conserved within the PKC family and has a catalytic region and regulatory region. The regulatory region has two main sub domains C1 and C2. Although several studies have investigated the role of the C1 domain, little is known about the function of the C2 domain, however there is some evidence that it acts as a protein interaction domain. PKCs are involved in a wide variety of cellular functions within cancer. PKC-δ has been demonstrated to have a particular involvement with the apoptotic processes of a cancer cell. A pro-apoptotic role for PKC-δ has been identified, whereby tyrosine phosphorylation of particular residues induces translocation to the nucleus, alongside a similar translocation event for caspase-3. In the nucleus, caspase-3 cleaves the regulatory and catalytic regions to form a free catalytic domain that is uninhibited by the regulatory portion. This free catalytic region causes the induction of the apoptotic pathway. Conversely an anti-apoptotic role has also been identified for PKC-δ. This was found in MDA-MB-231 cells, which have a ras mutation. Due to this mutation in these cells, ERK1/2 phosphorylation is high. Without PKC-δ activity the high phosphorylation levels induce apoptosis. PKC-δ acts on a pathway to reduce ERK1/2 phosphorylation, thus facilitating cell survival. This study aimed to investigate the role of the PKC-δ C2 domain within breast cancer cells. Through use of these techniques, the role of the C2 domain was examined in order to consider its utility as a drug target to treat breast cancer. •Constructs were developed of pIRESneo2 vector with myc-tagged C2 domain and myc-tagged PKC-δ sequences •Breast cancer cell lines, MDA-MB-468, MDA-MB-231 and MCF-7, were stably transfected with a vector control and myc-δC2 construct. MDA-MB-231 and MCF-7 were also transfected with myc-PKC-δ. •Cell lines developed were examined for alterations due to the presence of the C2 domain or PKC-δ over-expression As the C2 domain is proposed to be a protein interaction domain, we hypothesized that over-expression of this domain would interfere with endogenous PKC-δ interactions, through competitive inhibition, and thus we could identify C2 domain roles. The effect on the cells of the endogenous PKC-δ would be opposed by the C2 domain. Thus the role of endogenous PKC-δ could also be clarified for a particular situation - it would be the opposite of the effects induced by the myc-δC2 on the cells. In the MDA-MB-468 stable cell lines, immuno-fluorescence examination of the myc-δC2 cells showed the myc-δC2 was localised at the ends of actin protrusions from the bulk of the cell. The myc-δC2 expressing cells had a more extensive cytoskeleton than the Vector control cells, possibly suggesting improved attachment to a surface. An experiment examining this illustrated that the myc-δC2 cells appeared to attach in a shorter time period. This implies that the role of the endogenous PKC-δ is to discourage cell attachment and promote an invasive phenotype, this is in agreement with the literature. During sub-culture of the MDA-MB-468 cell lines it became apparent that the myc-δC2 cells were growing at an increased rate over the Vector cell lines. This was quantified and indeed the myc-δC2 cells did increase in cell number more than the Vector cells. This was also the case with the MDA-MB-231 cells but not with the MCF-7 cells. Growth is a balance of proliferation and apoptosis. This effect indicates that PKC-δ is pro-apoptotic, or anti-proliferative. MCF-7 cells lack caspase-3 and thus pro-apoptotic effects of PKC-δ would be affected in this cell line. As the myc-δC2 did not have an effect in these cells we examined apoptosis to see if these effects could be attributed to differences in apoptosis. The MDA-MB-468 cells expressing myc-δC2 had higher viability than the Vector cells. This fits with the cell number data, indicating that a lower level of apoptosis has led to a greater cell number, and advocates a pro-apoptotic role for PKC-δ. This was not the case in MDA-MB-231 cell lines where Vector cells had higher viability. This agrees with the literature describing PKC-δ displaying an anti-apoptotic role in this cell line, but does not fit with the cell number data. Thus, differences in cell number are likely due to effects on proliferation, although this was not investigated. MCF-7 cells showed no differences indicating the apoptotic program of these cells is indeed affected by the lack of caspase-3. Serum starvation is a commonly used method to induce apoptosis. MDA-MB-468 cell lines were serum starved in order to examine the effects. The apoptosis profile was altered and myc-δC2 cells showed lower viability than the Vector cells, indicative of an anti-apoptotic effect of PKC-δ. An anti-apoptotic effect is observed in MDA-MB-231 cells, where the effect was proteasome dependent. This was also the case in this situation. The anti-apoptotic effect is related to levels of phosphorylated ERK1/2, where high levels are pro-apoptotic. The phosphorylation status was examined and illustrated a much higher level of phosphorylation in myc-δC2 cells over Vector cells when starved. This indicates myc-δC2 is inhibiting the de-phosphorylating role of PKC-δ in these apoptotic cells. Thus it appears that the C2 domain acts as a ‘sensor’ to serum status, appropriating PKC-δ effects in apoptotic pathways according to the serum status, the method of which is unknown. This study has highlighted the importance of the PKC-δ C2 domain in breast cancer apoptosis. The effects appear to require a fully active PKC-δ pro-apoptotic pathway, and are dependent on the serum status of the cells. Further investigation would be required to identify a level of serum for use in vitro that is relevant to an in vivo tumour situation. If low levels are more relevant to a clinical state then it may be possible to target the C2 domain with drugs to allow induction of apoptosis. The differences between the cell lines clearly show that the phenotypic analysis of tumours would be vital to identifying whether such treatment would be applicable, as effects of any drug would vary greatly across tumour types. MDA-MB-468 and MDA-MB-231 are both triple negative cell lines (i.e. they do not express progesterone, oestrogen or HER2 receptors); however the strong differences seen in this case indicate further phenotypic analysis would be essential.

Mutations in proteolipid protein 1 (PLP1), an X-linked gene causes Pelizaeus-Merzbacher disease (PMD) in humans. The most frequent cause of PMD is the duplication of PLP1, which encodes the major myelin membrane protein of the human CNS. The #66 transgenic mice with extra copies of the wild type Plp1 gene are a valid model of PMD caused by increased gene dosage (Readhead et al., 1994). These mice develop dysmyelinating or demyelinating phenotypes dependant on the gene dosage. This study investigated the effects of both low and high increased Plp1 gene dosage on various different selected aspects of myelin, including morphology, message and protein levels of PLP/DM20 and other representative myelin proteins and PLP/DM20 dynamics. Early in development mice with low increased gene dosage (hemizygous) are indistinguishable at the protein and myelin levels from their wild type littermates. During myelination these animals display elevated levels of PLP/DM20 in the oligodendrocyte cell body and alterations in other myelin protein levels and to the structure of myelin but these are transitory effects. These transient changes suggest the oligodendrocytes at low gene dosage retain the ability to regulate expression, production and incorporation of proteins into myelin thus maintaining the normal process of myelination. At high increased gene dosage (homozygous), oligodendrocytes in culture, pre and early myelinating oligodendrocytes in vivo and oligodendrocytes in vivo during peak myelination all exhibit elevated levels of PLP/DM20 in the their cell bodies. The protein is sequestered into autophagic vacuoles and late endosomes/lysosomes (LE/Ls), while the levels in myelin are reduced compared to wild type and hemizygous cells. Synthesis, partitioning with lipids and incorporation of PLP/DM20 are all also affected in the homozygous animals. The increased Plp1 gene dosage does affect other myelin proteins, in particular MBP, which showed a consistent and dramatic reduction in oligodendrocytes and myelin. These results indicate the heterogeneity of phenotypes and underlying changes caused by low and high increased Plp1 gene dosage. The cause of the dysmyelination observed in #66 mice and patients with PMD does not appear to be due to one single change in myelinogenesis. Each alteration observed in #66 transgenic mice could be a contributing factor. Importantly, the perturbation of MBP expression, in the light of this gene’s pivotal role in myelination, highlights that the relationship between Plp1 and Mbp expression is implicated in the pathogenesis of dysmyelination.