Exposure to loud sound can cause a series of hearing problems, the most common being tinnitus or hearing loss (temporary or permanent). Furthermore, tinnitus caused due to acoustic trauma may be observed with or without hearing loss, making it harder for tinnitus researchers to understand the pathology of this condition. Despite extensive studies in both animal and human subjects, it is still not fully understood how acoustic trauma can change neuronal activity in the auditory cortex. Several animal studies suggest changes in auditory tuning properties and increase in spontaneous activity after exposure to acoustic trauma. However, there are several discrepancies in observed changes. One possible explanation for this could be that these findings represent an average response across cortical depths which could mask the layer specific alteration in neural activity following acoustic trauma because previous studies have shown laminar specific evoked and spontaneous activity. In this study we tested the hypothesis that acoustic trauma alters neural activity in a layer-specific manner. Rats were anesthetised with urethane anaesthesia and recordings were obtained using multichannel linear silicon probes inserted vertically into the primary auditory cortex. The animals were exposed (bilaterally) to one octave white noise centred at 16 kHz, at 110 dB SPL for 1 hour. Spontaneous and auditory-evoked activity was measured before trauma and then one and two hour time-points after the acoustic trauma. We quantified laminar specific and average changes in different tuning curve parameters such as threshold, characteristic frequency, bandwidth, sparseness, spontaneous firing rate and burst like activity after trauma exposure in three different frequency regions of primary auditory cortex. We observed laminar-specific changes in auditory tuning properties such as increase in threshold and spontaneous activity mainly in layer V of the primary auditory cortex following acoustic trauma. Furthermore, we also observed increase in burst-like spiking in the superficial layers. These findings support the hypothesis that acute effects of acoustic trauma on auditory cortical population activity is laminar-specific. These findings provide essential information regarding the changes in circuit mechanisms that develop following acoustic trauma which are critical for enhancing our knowledge about the pathology of these conditions and also to identify new potential targets to treat them.

Metabolomics can be used as an aid to functional genomics in order to investigate the functions of genes or enzymes. In the current study metabolomics was employed in the study of the response of LNCaP prostate cancer cells to sphingosine kinase inhibitors. Cell culture conditions, metabolite extraction and the LC/MS settings were optimized aiming at a reliable, unbiased, sensitive, and high throughput metabolomic protocol. Three different sphingosine kinase inhibitors were studied and reported in this work. A global metabolic profiling method based on electrospray ionisation mass spectrometry was developed for prostate cancer cells metabolites. The method involved optimizing the extraction of LNCaP cells metabolites followed by analysis using liquid chromatography coupled with high-resolution mass spectrometry (HRMS). Extraction repeatability and storage were studied and 480 metabolites were putatively identified. In the study protocol ~ 180 standard compounds from different chemic al classes were also run. Five different columns were compared in terms of their performance using these metabolites in combination with MS operated in both positive and negative electrospray ionization modes. The ZIC-pHILIC column showed the best performance and the highest number of metabolites separated. An effect of storage conditions on metabolite profiles was assessed using multivariate statistics (PCA). The treatment of LNCaP and LNCaP-AI cells with 2-(p-hydroxyanilino)- 4-(p-chlorophenyl)thiazole (Ski) modulated the metabolome, with marked changes in glutathione, NADPH, pentose phosphate shunt and glycolytic metabolite levels which were indicative of a pronounced oxidative stress response and modulation of the Warburg effect. Diadenosine triphosphate (Ap3A) was not detected in LNCaP-AI but was present in LNCaP. Ap3A and diadenosine tetraphosphate (Ap4A) are novel apoptotic markers and were quantified by using tandem mass spectrometry.(R)-FTY720 methyl ether (ROME), which is a SK2-selective inhibitor, did not affect produce oxidative stress or affect the pentose phosphate pathway but increased in the levels of several lysophosphatidylinositols (Lyso PI). However, increases in phosphatidylserine (PS), sphingosine and sphinganine, hydroxysphingosine and hydroxysphinganine were marked when the cells treated with (S)-FTY720 Vinylphosphonate. In addition, it caused a fall in hypoxanthine, guanine and uridine which which may be linked with purine nucleoside phosphorylase (PNP). Cell based metabolomics provides a method for exploring the mechanism of drug action.

This thesis describes the activity of selected plants against biofilms of Pseudomonas aeruginosa strain PA14. The activity of plant extracts and subsequently purified compounds was evaluated using a stepwise separation process called bioassay-guided fractionation and used a microtitre plate based assay. Active extracts (showing more than 50 % biofilm inhibition, BFI) were further investigated for the presence of active compounds. The fractionation process involved the use of chromatographic techniques. Compounds were identified using NMR, GC-MS and LC-MS. Out of a total 129 extracts screened for antibiofilm activity using microtitre plate method, 44 extracts showed more than 50 % biofilm inhibition, whilst 85 extracts were found to increase biofilm formation. Four active extracts, (E333), (E341), (H338) and (M338) were selected for further investigation. A process of bioassay-guided fractionation was used to purify the phytochemicals present in each active extracts. This study led to the discovery of four bioactive compounds, namely (E333F1S1), (E341), (HA6) and (M338B) with antibiofilm activity against P. aeruginosa PA14. The active fraction (E333F1S1) from Ribes nigrum leaf was found to contain mixtures of alkanes such as n-nonadecane, 2-methylnonadecane, 2-methylicosane and 2 -methyloctacosane. The active extract of Sambucus nigra flower (E341) contained a mixture (2:1 ratio) of oleanolic and ursolic acid. Comparable activity was found at the same ratio (2:1) when these compounds were tested as a mixture of pure compounds. No significant difference (p < 0.05) in activity compared to a positive control was observed when these compounds were combined in different ratios. Only weak antibiofilm activity was observed when each compound was tested on its own. The LC-MS analysis on Coriandrum sativum seed active fractions, (HA6) revealed the presence of putative compounds such as 10-undecenal, dodecanal, 2-hexyl furan, linalool oxide, caryophyllene oxide and 4-ethylcamphor. When linalool oxide was tested, it exhibited a strong activity but reduced activity when in mixtures. Both activities were significantly different (p < 0.05) compared to positive control. Another active fraction of Coriandrum sativum seed, (M338B) showed the presence of putative compounds such as fatty acids, carboxylic acid, carboxylate and tetraone. This study led to the discovery of potential bioactive compounds from selected plant as antibiofilm agents against P. aeruginosa PA14. The bioactive compounds were from Ribes nigrum leaf (e.g. mixture of alkanes), Sambucus nigra flowers (e.g. mixture of ursolic acid and oleanolic acid) and Coriandrum sativum seeds (e.g. mixture of oxygenated monoterpenes, carboxylic acid, carboxylate, tetraone, glycerol, carbohydrates and fatty acids).

Drug discovery from natural sources including plants, marine organisms and endophytes is still pivotal in pharmaceutical research due to their ability of producing diverse and unique secondary metabolites. Discovery of secondary metabolites from leaves of Vitex pinnata, a medicinal plant from Malaysia and its associated endophytic fungi, Lasiodiplodia theobromae, Nigrospora sp and Pestalotiopsis olivacea afforded nineteen compounds and several of these compounds exhibited good anticancer, antitrypanosomal and anti-mycobacterial activities. Secondary metabolites isolated from leaves of V. pinnata previously reported to exhibit good anti-inflammatory activity and interestingly the leaves were used by local Malay community to treat cuts and wounds. In recent years new strategy using metabolomics in natural products is trending among scientists and found to be efficient, intelligent and robust. Implements of metabolomics including hyphenated HR-LCMS, NMR and integrated with in-house database, AntiMarin were utilised to exploit endophytic fungal natural products from V. pinnata. Preliminary exploration of secondary metabolites production of all three endophytic fungi was simply achieved by applying metabolomics strategy for medium-scale fermentation. Metabolomics also was used as decision-making strategy in mining active metabolomes of endophytic fungus, Lasiodiplodia theobromae against Trypanosoma brucei brucei. Dereplication approach using metabolomics for identification of compounds from Pestalotiopsis olivacea was easily accomplished.

Cisplatin iscurrently aleading anticancer drug used in the treatment of various cancers. Its clinical use, however, is limited by its poor bioavailability, its undesirable toxic side effects profile and by the ability of certain tumours to develop cisplatin resistance. A method that may overcome these problems is the reversible encapsulation of cisplatin within the cavity of a macro cyclic container such as cucurbit[7]uril; a macrocycle formed by the acid condensation of glycoluril and formaldehyde, which has been shown to overcome acquired resistance in an in vivo human tumour xenograft model via a pharmacokinetic effect. In the first section of this thesis an implantable hydrogel based system was developed for the delivery of cisplatin and cisplatin@CB[7]. First, cisplatin was encapsulated within cucucrbit[7]uril (CB[7]) to form the host-guest complex: cisplatin@CB[7]. This was then incorporated into gelatin and 0-4% w/v polyvinyl alcohol (PVA)-based hydrogels as slow release drug delivery systems. In vitro studies of the hydrogels demonstrated predictable yet not significant swelling and disintegration dependent on their PVA concentration. The hydrogel with the highest PVA content was slower to swell and release drug compared with hydrogels containing lower concentrations of PVA. The effect of the hydrogel's PVA concentration on in vitro cytotoxicity was examined using A2780/CP70 ovarian cancer cells with results showing a significant reduction in cytotoxicity as the hydrogel's PVA concentration increased which indicated that a slow release system was achieved. Over the 24 hours of drug exposure time used, hydrogels containing 4% PVA loaded with 1mM of cisplatin@CB[7] showed a 19 ± 0.01% (p = 0.004) decrease in viable cells compared to the control, whereas hydrogels containing 0% and 2% PVA induced an 81.2 ± 0.003% (p = 0.0005) and 42 ± 0.02% (p = 0.0002) inhibition of cell growth, respectively. Finally, the in vivo efficacy of a 2% PVA hydrogel implanted under the skin of nude mice bearing A2780/CP70 xenografts showed that low dose hydrogels containing cisplatin@CB[7] (30 μg equivalent of drug) was just as effective as an intraperitoneal high dose administration of free cisplatin (150 μg) at inhibiting tumour growth. Overall, the results suggest an ability of implantable hydrogels to treat cancers with much lower doses of drug, thereby reducing the severity of the toxic side effects induced. In the second section of this thesis, cisplatin, CB[7] and cisplatin@CB[7] were tested and compared for their ex vivo neurotoxic, neuromyopathic and cardiotoxic activity amongst other platinum-based drugs (K₂PtCl₄, 56MESS and PHENEN) and macrocyclic drug delivery systems (CB[6], β-cyclodextrin, Motor2 and pentamer) using electrophysiological methods. The neurotoxic activity of the drugs was studied using mouse desheathed sciatic nerve preparations. Both cisplatin and CB[7] administered at a concentration of 300 μM decreased the amplitude of the nerve compound action potential (nCAP) by 13 ± 4.7% ( p = 0.3) and 4 ± 0.2% ( p = 0.8), respectively over an 80 minute period. Neuromyopathic activity was studied using the chick biventer cervicis nerve muscle preparation. Results showed that incubation of the nerve-muscle tissue with 300 μM of cisplatin caused statistically significant muscle paralysis to occur by decreasing the electrically stimulated muscle twitch response by 96 ± 4% (p = 0.001), through interference in the presynaptic neuron. Whereas, CB[7] caused a statistically significant muscle paralysis by decreasing the electrically stimulated muscle twitch response by 84 ± 9% (p = 0.001) through interference with the postsynaptic muscle membrane. Cardiotoxic activity was examined using the rat right and left heart atria. Results show that incubation of the atria tissue with 300 μM of cisplatin reduced the contraction rate of the right atria by 68.8 ± 1% (p = 0.001) and in the left atria by 1 ± 1% (p = 0.4) by the end of the two hour period study. When incubated with 300 μM of CB[7], the contraction rate in the right atria increased by 31 ± 13.6% (p = 0.3) and decreased in the left atria by 10 ± 3.5% (p = 0.3) Finally, the effect of the encapsulation of cisplatin by CB[7] on its neurotoxic, neuromyopathic and cardiotoxic activity was investigated. Results show that while the encapsulation had no effect on the neurotoxic activity of cisplatin, the encapsulated complex reduced the extent of cisplatin's neuromyopathic activity by reducing the muscle paralysis induced by cisplatin by 60%. When encapsulated by CB[7], the cardiotoxicity of cisplatin on the contraction rate of the right atria was also significantly decreased from 65.8% to 11%. In conclusion, these results suggest that CB[7] could exhibit neuromyopathy and cardio protective properties as it reduced the neuromyopathic and cardiotoxic activity of the encapsulated cisplatin.

Continuous processing is a widely used application that provides a number of benefits to manufacturing including improved process control and high quality products. The continuous oscillatory baffled crystalliser (COBC) is a type of continuous platform that enables crystallisation and growth of pharmaceutical systems, moving crystallising solutions and suspensions through a series of baffled tubes. Application of oscillatory flow results in the formation of eddy currents enabling an efficient mixing and near-plug net flow experience of the bulk solution. The enhanced turbulence provides the possibility of linear scale up and uniformity in bulk solution environment (distributions of shear rates and temperature gradients are reduced) alongside the decoupling of mixing and net flow resulting in long residence times. Considering this, COBC systems offer promise to the enable effective operation and scaling of pharmaceutical crystallisation to industrial-sized processes. Residence time distributions (RTD) in a DN15 COBC have been studied for the first time as a function of flow and oscillatory conditions, illustrating operation with a near-plug profile. A novel moving fluid (MF) batch oscillatory baffled crystalliser has been developed as an improved model of the hydrodynamic conditions in a DN15 COBC and thus eliminates many of the assumptions made during the tradition approach for moving to continuous oscillatory flow. This batch system was combined with imaging technology to investigate nucleation and fouling. Experiments illustrated variations in nucleation kinetics for L-glutamic acid polymorphs when comparted to the tradition batch platforms, thus highlighting the importance to use representative systems for crystallisation scale-up. Polymorph control during continuous oscillatory crystallisation was investigated using two pharmaceutically relevant systems: L-Glutamic Acid and Carbamazepine. Due to excessive fouling in the COBC during spontaneous nucleation, continuous processes were designed and operated to decouple the two step crystallisation process. Through the use of seeding, fouling could be eliminated, and a focus on the control over particle growth could be made. In addition, a basic methodology from batch to continuous oscillatory baffled crystallisation is presented to achieve polymorphic control. This work has advanced the practical and scientific understanding towards a methodology for successful oscillatory flow operation in addition to the possibilities of a control approach, through implementation of process analytical technologies, thus facilitating the rapid development and understanding of COBC processes in future studies.

The present study focuses on a series of physiological studies to study oxidative and nitrosative stress and online monitoring of fungal biomass growth in submerged batch cultures of the filamentous fungus Aspergillus niger B1-D, modified to secrete lysozyme. The results of the undertaken studies described here show that increasing concentrations of polymyxin B, added to a bioreactor, increase oxidative stress during cultivation of Aspergillus niger B1-D in a bioreactor. This was shown by the increased superoxide dismutase (SOD) and catalase (CAT) activities after addition of polymyxin B. This increased oxidative stress also results in a decrease in production of lysozyme, which is at least 75% lower in all polymyxin B processes. Varying oxygen transfer rate (OTR), by changing the stirring rate, in the lag phase of a batch fermentation, utilising the Aspergillus niger B1-D strain, results in increased oxidative stress with increased stirring rates. The resulting lysozyme production was at least 75% lower compared to the optimum agitation setting. This optimum setting was 200-400 rpm, which was controlled by the dissolved oxygen tension. Adding increasing amounts of sodium nitroprusside (SNP) reduces oxidative stress resulting in an increased production of lysozyme. The use of SNP also showed increased CAT activities, which points towards an interaction between oxidative and nitrosative stress due to the release of nitric oxide from SNP. Applying an online biomass sensor (Buglab) during batch cultivation of Aspergillus niger B1-D showed that this sensor can accurately monitor fungal biomass growth from 7 g/l onwards.

Leishmaniasis is a public health problem and development of a vaccine to prevent infection is required. The overall aim of this study was to develop a vaccine to protect against Leishmania infection using L. donovani, L. mexicana and L. major gamma glutamylcysteine synthetase (γGCS) recombinant proteins. Studies to optimise the expression of L. donovani, L. mexicana and L. major γGCS recombinant proteins showed that induction with 0.1 mM isopropyl β-D-1-thiogalactopyranoside produced the highest amounts of recombinant protein, and incubation of bacteria at 18°C after induction increased the amount of soluble protein produced. Recombinant L. mexicana γGCS had significantly higher specific enzyme activity (p <0.001) compared to L. donovani and L. major γGCS. L. mexicana γGCS was the most resistant to L-buthionine sulphoximine (BSO) inhibition, the specific irreversible inhibitor of γGCS, at the maximum concentration tested (1.5 mM) and L. mexicana promastigotes were the most resistant to the cytotoxic effects of BSO compared to L. donovani and L. major promastigotes (p < 0.001). Vaccination with the recombinant γGCS proteins from L. donovani, L. major and L. mexicana (triple vaccine) induced significant parasite-specific Th1 and Th2 immune responses based on antibody titres and cytokine production by in vitro stimulated splenocytes from immunised mice. Vaccination by inhalation or subcutaneous injection with the triple vaccine was similar mean percentage reduction in parasite burdens compared to controls ± SE, was 98% ± 0.02 in L. mexicana infected mice. In L. major infected mice was 70% ± 0.1 by subcutaneous immunisation and 65% ± 0.01 for inhalation vaccination. Treatment with the triple vaccine by inhalation failed to protect mice against L. donovani infection but was effective in hamsters, where a significant reduction in liver and bone marrow parasite burdens compared to control values (p < 0.05; mean percentage reduction compared to controls ± SE: spleen 89 ± 1; liver 83 ± 0.3; bone marrow 77 ± 1). In conclusion, the results of this study indicate that vaccination against leishmaniasis is feasible by the pulmonary route and that the triple vaccine is a potential vaccine candidate.

Proteinase-activated receptors (PARs) are a family of novel G protein-coupled receptors, which are widely expressed in the brain. It has been recently shown that PAR2 activation indirectly modulates hippocampal neuronal excitability and synaptic transmission in vitro, and treatment with the PAR2 agonist SLIGRL-NH2 induced deficits in tests of motivational learning in rats. Hence, in this study we have investigated whether PAR2 deletion affects mouse behaviour in tests of learning and emotional behaviours under physiological and pathological conditions. Deletion of PAR2 had no effect on locomotor activity in the open field test. Heterozygous males appeared more anxious in the open field test but knockout females exhibited less anxiety in the elevated plus maze. PAR2 deletion had no effect on spatial reference memory in the Morris water maze but reduced mean percent alternation to chance level in the T-maze continuous alternation test and produced deficits in the male mice in the novel object recognition test. Deletion of PAR2 did not affect general startle reactivity and sensorimotor gating but it decreased the startle response at the highest stimuli in females. In a sickness behaviour model, deletion of PAR2 delayed induction of anhedonia as measured in the sucrose preference test after injection of lipopolysaccharide. It also induced a more rapid recovery from other symptoms of sickness behaviour as shown by increased locomotor activity 24 and 48 h post injection, increased body weight at 48 and 72 h post injection, increased food intake during the second day post injection and reduced anxiety-like behaviour 24 h post injection. The novel PAR2 agonist AC-264613 penetrated into mouse brain. AC-264613 had no effects on locomotor activity and anxiety-like behaviour but showed a tendency to induce anhedonia. In conclusion, these data indicate that proteinase-activated receptor-2 may be involved in mouse behaviour under normal and pathological conditions.

Schizophrenia is a complex and debilitating psychiatric disorder. Dysfunction of the NMDA subtype of glutamate receptors is implicated in deficits found in schizophrenia, and some of these deficits may be reproduced in rodents using the NMDA receptor antagonist, phencyclidine (PCP). Nitric oxide synthase (NOS) is the synthesising enzyme of the gaseous neuromodulator, nitric oxide. The neuronal isoform of NOS (nNOS) is functionally associated with NMDA receptors. The role of NOS in schizophrenia is not fully understood. The primary aim of this thesis is to investigate NOS signalling in cultured neurones and in rodent PCP models of relevance to schizophrenia. Using diaminofluorescein microscopy of primary neuronal cultures, it is shown that non-selective inhibition of NOS using L-NAME, and selective inhibition of nNOS using L-NPA reversed glutamate-stimulated nitric oxide generation in hippocampal and cerebellar neurones, but inhibition of endothelial NOS using L-NIO did not. Novel c ompounds that modulate the NOS cofactor, tetrahydrobiopterin, altered nitric oxide generation in cerebellar neurones. NOS activity was increased in the hippocampus, and decreased in the reticular thalamus in mice administered acute PCP (5 mg.kg-1, i.p.), as determined by NADPH-diaphorase activity. NOS activity normalised in these areas with subchronic PCP (5 mg.kg-1 twice daily for 5 days), and NOS activity was decreased in the prefrontal cortex. Decreased activity of thioredoxin reductase was found in the hippocampus and thalamus with acute PCP, but was unchanged with subchronic PCP. Pretreatment with L-NAME (40 mg.kg-1) and L-NIO (20 mg.kg-1) improved hyperlocomotion and III deficits in prepulse inhibition observed with PCP, but L-NPA (20 mg.kg-1) did not. In conclusion, the results presented in this thesis give evidence for a role of NOS in deficits observed in rodent PCP models of relevance to schizophrenia. Selective inhibition of NOS isoforms is a potential therapeutic strategy to improve deficits associated with NMDA receptor dysfunction found in schizophrenia.