Apoptosis studies demonstrated the biphasic pro- and anti-apoptotic effects of pure NO donors, SPER/NO and DEA/NO, and the pro-apoptotic effects of ONOO- donors, GEA 3162 and SIN-1, in human neutrophils. Low concentrations of the pure NO donors delayed the rate of neutrophils apoptosis, while high concentrations of all compounds tested accelerated cell death. Time course analyses of four independent events of apoptosis revealed that morphological and biochemical parameters of neutrophils apoptosis may proceed independently of internucleosomal DNA fragmentation, which has long been considered a key hallmark of apoptosis and is frequently used as the sole criterion for assessment of this form of cell death. Treatment with high concentrations of ONOO- donors and, to a lesser extent, with the longer-lasting pure NO donor, SPER/NO, induced morphological and cell surface (CD16 shedding and phosphatidylserine exposure) changes characteristic of neutrophils apoptosis, but paradoxically inhibited internucleosomal DNA fragmentation, as measured by propidium iodide intercalation and gel electrophoresis. In contrast, treatment with the short-lasting NO donor, DEA/NO, produced no such inhibition. An oxidation reaction was shown to be responsible for the suppression of the DNA fragmentation pathway, as the reducing agent, dithiothreitol, restored DNA fragmentation back to control levels. However, GEA 3162-mediated inhibition of DNA fragmentation did not occur upstream or at the level of degradation of inhibitor of caspase-activated DNase (ICAD/DFF45). Therefore, NO can exert either pro- or anti-apoptotic effects on human neutrophils apoptosis, depending on its concentration and flux, whereas only pro-apoptotic effects are achieved with ONOO- donors. Cell death promoted by ONOO- or SPER/NO is independent of an increase in internucleosomal DNA fragmentation. Thus, NO and ONOO- are able to modulate the rate of neutrophil apoptosis, which may have implications for chronic inflammatory conditions.

Assessment of sustained attention in rodents can be performed using the 5-choice serial reaction-time (5-CSR) task; analogous to the continuous performance test used in man. A 5-CSR protocol was established which allowed the demonstration of nicotine-induced improvements in sustained attention in mice. In this task α7 nAChR knockout (KO) mice exhibited impaired acquisition and performance, providing additional evidence that this receptor may be a valid therapeutic target for cognitive enhancement. In order to investigate the role of nAChR manipulation on working memory, the odour span task, a test of olfactory working memory capacity, was established in mice. Nicotine administration did not improve performance of C57B1/6J mice probably as a consequence of ceiling effects. Transgenic mice over-expressing human caspase-3 (hc-3) displayed a robust impairment in the task that was attenuated by nicotine administration. Moreover α7 nAChR KO mice exhibited impaired acquisition and performance in the task but in a different pattern to that of the hc-3 mice. This pattern may reflect an impaired ability to attend to the task as opposed to a working memory deficit alone. These demonstrations provide further support for a role of the α7 nAChR in cognition. Tg2576 mice represent the best well characterised transgenic model of AD, however there remains a dearth of information on their attentional and olfactory capabilities. The mice exhibited a deficit in sustained attention in the 5-CSR task, as well as an age-related impairment in the odour span task. In conclusion the development of the 5-CSR task for mice was used to identify a nicotine-induced improvement in normal mice and impaired performance in α7 KO and Tg2576 mice. In summary these data provide some evidence for a role of the α7 nAChR in nicotine-induced improvement in cognition, and with the tasks developed provide new tools for the assessment of putative cognitive enhancing compounds.

No description available.

Industrial manufacturing processes for pharmaceutical products require a high level of understanding and control to demonstrate that the final product will be of the required quality to be taken by the patient. A large amount of data is typically collected throughout manufacture from sensors located around reaction vessels. This data has the potential to provide a significant amount of information about the variation inherent within the process and how it impacts on product quality. However to make use of the data, appropriate statistical methods are required to extract the information that is contained. Industrial process data presents a number of challenges, including large quantities, variable sampling rates, process noise and non-linear relationships. The aim of this thesis is to investigate, develop and apply statistical methodologies to data collected from the manufacture of active pharmaceutical ingredients (API), to increase the level of process and product understanding and to identify potential areas for improvement. Individual case studies are presented of investigations into API manufacture. The first considers prediction methods to estimate the drying times of a batch process using data collected early in the process. Good predictions were achieved by selecting a small number of variables as inputs, rather than data collected throughout the process. A further study considers the particle size distribution (PSD) of a product. Multivariate analysis techniques proved efficient at summarising the PSD data, to provide an understanding of the sources of variation and highlight the difference between two processing plants. Process capability indices (PCIs) are an informative tool to estimate the risk of a process failing a specification limit. PCIs are assessed and developed to be applied to data that does not follow a standard normal distribution. Calculating the capability from the percentiles of the data or the proportion of data outside of the specification limits has the potential to generate information about the capability of the process. Finally, the application of Bayesian statistical methods in pharmaceutical process development are investigated, including experimental design, process validation and process capability. A novel Bayesian method is developed to sequentially calculate the process capability when data is collected in blocks over time, thereby reducing the level of noise caused by small sample sizes.

Background & Objectives: The potential benefits of adjuvant pharmacotherapy with N-acetylcysteine and acetyl-L-carnitine to reduce the amount of primary sensory neurons in the dorsal root ganglion (DRG) which die by apoptosis after peripheral nerve injury has been previously established. However, the duration of NAC therapy sufficient to achieve neuroprotection is unknown. Establishment of clinical trials of NAC/ALCAR therapy is hindered by relatively poor existing methods of assessment of neuronal populations and nerve regeneration in human subjects. Magnetic Resonance Imaging (MRI), a non-invasive modality, has been demonstrated in a pilot study to provide volumetric analysis of rat L4 DRG after sciatic nerve transection; detecting DRG volume reductions which reflect neuronal loss. Further, MRI has been reported to detect changes occurring in peripheral nerves distal to the site of injury, corresponding to axonal degeneration and subsequent regeneration. High Frequency Ultrasound (HFUS) is a new, high resolution imaging modality, not previously applied to the study of animal or human peripheral nervous system. Experiments were designed to test the duration of NAC treatment needed to achieve a neuroprotectant effect; to further evaluate the MRI DRG imaging protocol under interventional experimental conditions and in vivo; to investigate the ability of MRI and HFUS to visualise murine sciatic nerve and its response to injury and to assess the ability of HFUS to visualise human digital nerves in the hand. Further, a pilot study investigating the effect of NAC and ALCAR on primary sensory neurons in tissue culture was carried out to assess the neurotrophic effect of these agents in vitro. Methods: 1. Groups of rats underwent sciatic nerve transection, and NAC treatment for 7, 14, 30 or 60 days. At 60 days post injury neuron counts from L4 and L5 DRG were assessed histologically using a stereological optical fractionator analysis, and compared to the contralateral noN-axotomised DRG. 2. Animals from treatment groups were MRI scanned after two months to measure the L4 DRG volume bilaterally. A single animal was also scanned under anæsthesia and after death. 3. Numerous MRI techniques were used to scan rats after sciatic nerve crush, division and intraneural injection of contrast, attempting to identify the nerve and its response to injury. 4. Normal and injured rat sciatic nerve and intact human digital nerve were assessed by HFUS. 5. Cultured DRG neurons were exposed to a range of ALCAR/NAC doses or NGF, with assessment of cell survival and neurite formation at 24h. Results: 1. Neuronal death was related to duration of NAC treatment; with losses of 20%, 9%, 4% & 0% after treatment for 7, 14, 30 and 60 days respectively. 2. MR measured L4 DRG volume reduction correlated well with neuron loss. In vivo MRI scanning is feasible; however images are subject to motion artefact distortion, precluding accurate quantification. 3. The rat sciatic nerve could not reliably and consistently be identified by MRI in this model. 4. High frequency ultrasound can image sciatic nerve and its branches and human digital nerve with good resolution and can detect neurotmesis and axonotmesis grade injurys. 5. Non-toxic in vitro NAC/ALCAR dosage regimens have been defined, but do not affect neuron survival or neuritogenesis in the absence of NGF. Conclusions: 1. One month treatment with NAC post neurotomy prevents death of the majority of axotomised neurons. Apoptosis is prevented rather than delayed for some neuronal populations by NAC treatment. 2. MRI is a valuable objective tool, and DRG volume serves as a proxy measure of neuronal loss, fit for translation to human studies. 3. In vivo MRI DRG imaging requires motion compensation techniques such as respiratory gaiting. 4. Further investigation is needed to facilitate murine sciatic nerve imaging with MRI in this model, but HFUS easily demonstrates two grades of peripheral nerve injury, and is proposed for investigation of human digital nerve trauma. 5. The neuroprotective and regenerative effects of NAC/ALCAR on primary sensory neurons in vivo can not be replicated by direct treatment of isolated neurons in culture.

Biphenyl metabolism has been extensively studied in isolated viable adult rat hepatocytes in suspension with regard to both phase I and phase II reactions. A wide range of primary and secondary metabolites are produced by these cells, which closely reflects the situation appertaining vivo. Using biphenyl, and the three primary metabolites, 2-, 3- and 4-hydxoxybiphenyl, assessment of toxicity due to the substrate, primary or secondary metabolites, examination of the relationship between phase I and phase II metabolism, and the effect of inducers and inhibitors on the total metabolic profile has been made. Possible rate limiting phenomena operating in the intact cell that affect the rate of xenobiotic metabolism have also been studied. With this insight into the isolated hepatocytes metabolic capabilities, particularly with respect to xenobiotic metabolism, isolated adult rat hepatocytes in suspension and primary maintenance Culture were then used as to vitro model systems for the assessment of xenobiotic-induced toxicity. Using a mixed liver cell approach, the fibroblast cytotoxicity (as measured by inhibition of cell growth) of a number of xenobiotics is shown to be fully expressed only when metabolised to their 'active' species by the hepatocytes to vitro, closely reflecting the situation known to occur to vivo. The versatility and general applicability of such a mixed-cell approach, to in vitro toxicity and carcinogenicity assessment of xenobiotics is discussed. In order to decide whether hepatocytes model systems could be used to assess a xenobiotics hepatotoxic potential, the changes in viability and functional capabilities of cultured hepatocytes were monitored after exposure to known in vivo hepatotoxic agents. Results presented here indicate that xenobiotics that are hepatotoxic in vivo are also hepatotoxic vitro, though whether by a similar mechanism is as yet unclear. The advantages and limitations to the use of such in vitro hepatocyte model systems for the assessment of xenobiotic-induced toxicity in the light of the wide range and variety of other in vitro models that are currently being used are discussed.

The 9 + 2 microtubule axoneme of flagella and cilia represents one of the most iconic structures built by eukaryotic cells and organisms. Some cilia are motile, whilst others serve as sensory organelles, and can variously possess 9+2, 9+0 axonemes and other associated structures. The recent emergence of ciliary dysfunction as a cause of human disease has renewed interest in these organelles, with several genomic and proteomic studies of cilia and flagella now published from various systems. I identified ten genes from such screens, and functionally characterised them in the flagellate protozoan parasite Trypanosoma brucei. One further gene was identified by searching a gene locus for polycystic kidney disease, a disease linked to malfunction of the primary cilium. I carried out a bioinformatic screen on the selected genes to determine their phylogenetic distribution. Cell lines expressing GFP fusion proteins were made to assess the localisation of this set of proteins, which were found to localise to a range of flagellar compartments including the axoneme and regions of the basal body. The well established inducible RNA interference system in T. brucei was then used to assess function of this set of proteins. In procyclic form trypanosomes the ablation of five proteins produced motility phenotypes when ablated; in some, but not all, this was accompanied by defects in axonemal ultrastructure. Two of these are closely related homologues and showed functional redundancy; when ablated independently there was no difference in motility, however simultaneous ablation produced flagellar paralysis. RNAi-mediated ablation of proteins affecting flagellar motility in bloodstream form trypanosomes produced rapid cytokinesis defects, suggesting that impairment of flagellar function may provide a novel avenue for disease control.

Eicosapentaenoic acid (ERA) is an w-3 polyunsaturated fatty acid (PUFA) with anti-inflammatory and anti-cachetic properties. These properties have not previously been investigated simultaneously with regards to skeletal muscle damage and regeneration and therefore the main aim of this work was to examine the possible protective effects of ERA during damage induced by the pro-inflammatory cytokines TNF-a and TWEAK . Using an established model of myoblast differentiation, skeletal muscle cell differentiation was shown to be morphologically impaired by TNF-a and TWEAK. Formation of myotubes was restricted as shown by reduced myoblast fusion indices (Ml) and myotube widths. These effects were associated with TNF-a induced apoptosis. Both cytokines caused increased expression of pro-inflammatory gene targets of NF-KB activity; TNF-a and IL- 6 and in parallel, TNF-a treatment increased NF-KB activity and inhibited expression and activation of PPARy. EPA prevented the TWEAK /TNF-mediated loss of expression MyHC expression (reduced to 5-30% Ml) and significantly increased myogenic fusion (p<0.05) and myotube diameter (p<0.05) indices back to control levels (Back to approx 70% Ml). ERA protective activity was associated with blocking cell death pathways as ERA completely attenuated TNF-mediated increases in caspase-8 activity (p<0.05) and cellular necrosis/apoptosis (p<0.05) back to their respective control levels. This led to investigation of the possible mechanisms of action for ERA using the TNF-a damage model. ERA blocked NFKB activation, downstream gene targets of NF-KB activity, IL-6 and TNF-a, and was associated with upregulation and activation of PPARv. Pre-treatment with a specific PPARy antagonist (GW9662) inhibited these actions of ERA, thus confirming that ERA activity was at least partially dependent on PPARy. This thesis has shown for the first time that PUFA such as ERA promote skeletal muscle differentiation in opposition to pathological levels of inflammatory cytokines. Thus, PUFAs such as ERA may represent a class of naturally occurring, low toxicity, PPAR ligands that could have clinical applications for treatment of inflammation-associated ongoing skeletal muscle damage in chronic disease states or ageing.

N-myristoyltransferase (NMT), a ubiquitous enzyme, found in a number of organisms including fungi, parasites and humans, catalyses the irreversible transfer of a C-14 chain (myristic acid) to the N-terminal glycine of specific target proteins, a process known as N-myristoylation. NMT has been reported to be essential for the viability of parasites such as Trypanosoma brucei and Leishmania donovani which are responsible for the diseases African sleeping sickness and Leishmaniasis respectively. This suggests that NMT is a promising target for antiparasitic drug development. The work detailed in this thesis describes the use of a piggy-back approach in the design and synthesis of peptidomimetic NMT inhibitors derived from lead compounds originally reported as antifungal NMT inhibitors. NMT, in this report, is explored as a target for the study of malaria and leishmaniasis which are caused by Plasmodium and Leishmania respectively. The current therapeutic treatments of these two diseases, the role of N-myristoylation in the parasitic organisms will be discussed; this thesis will also detail the ligand- and structure-based synthesis of NMT inhibitors using solid phase peptide synthesis. The synthesised inhibitors were tested against P. vivax (PvNMT), P.falciparum (PfNMT), L. donovani (LdNMT) and human enzyme Homo sapiens NMT1 (HsNMT1) using a fluorogenic assay to generate an expansive structure-activity relationship (SAR). A structure- guided methodology using structural information from high resolution crystal structures in both Plasmodium and Leishmania was also employed to rationally design inhibitors. L.majorNMT (LmNMT) was used, for the first time, in a fragment-based screen and 9 hits were attained. Crystal structures of four of the fragments in the library were successfully generated in LmNMT. This work led to the discovery of potent peptidomimetic inhibitors of P. vivax NMT and L. donovani which were design ed from antifungal inhibitors, thus validat ing the piggy-back approach in the study of NMT as a target for malaria and leishmaniasis.

This thesis is composed of two separate projects: Kinetic Template-Guided Tethering of Fragments and Design and Synthesis of a Chemical Probe to Dissect the Cellular Signalling Cascade leading to Cyclin D1 Degradation after DNA Damage. Kinetic Template-Guided Tethering of Fragments The development of a novel methodology for the site-directed discovery of small molecule, protein-binding ligands is described. The protein of interest, with a cysteine thiol (either native or engineered) adjacent to the desired binding pocket, is incubated with mixtures of low molecular weight compounds (fragments) modified with either an acrylamide or a vinyl sulfonamide capture group. Any ligand within the mixture that binds within the pocket brings the capture group into close proximity with the cysteine thiol, promoting a conjugate addition reaction at an increased rate over the background reaction. The capture reaction is designed to be slow, such that during the time course of an experiment, no adduct formation is observed unless the reaction is templated by the protein. By this method, binding ligands are rapidly identified by mass spectrometry analysis of the crude reaction mixtures. The methodology has been termed 'kinetic template-guided tethering'. Design and Synthesis of a Chemical Probe to Dissect the Cellular Signalling Cascade leading to Cyclin D1 Degradation after DNA Damage An inhibitor described within the literature was found to attenuate the reduction of cyclin D1 after DNA damage to cells. In order to implement a two-step chemical proteomics strategy to find the molecular target of this inhibitor, a synthesis of the compound with an alkyne appendage was required. The alkyne acts as a functional handle for attachment of a reporter molecule in cells via the Huisgen cycloaddition ('Click') reaction. The design and synthesis of this inhibitor with the alkyne appendage is described.