Neurosteroids are recognized as important modulators of functioning of the nervous system. They have been recognized to influence several neurobehavioral processes including memory and cognition. Lindane, an organochlorine pesticide has been shown to adversely affect memory and induce oxidative stress on both acute and chronic exposure. The present study was thus designed to explore the modulation of effects of lindane over cognitive function by progesterone (PROG), pregnenolone sulfate (PREG-S) and 4’-chlorodiazepam (4CD).Male Wistar rats were used for the study. The control groups were administered a) the vehicle in which the drugs were dissolved b) lindane and c) PROG, PREG-S or 4CD alone. The other three groups received PROG, PREG-S or 4CD following pre treatment with lindane for a period of 6 weeks. Cognitive function was assessed using step-down latency (SDL) on a passive avoidance apparatus and transfer latency (TL) on a plus maze weekly starting from one day before beginning of any treatment till the end of study period. Oxidative stress was assessed at the end of the study period by examining brain malondialdehyde (MDA) and reduced glutathione (GSH) levels.1.Control group showed no change in cognitive function during the study period.2.The group treated with lindane showed a decline in cognitive function as measured by increased TL and decreased SDL. This group also showed significant a increase in oxidative stress as witnessed by an increase in brain MDA and decrease in brain GSH levels.3.PROG), PREG-S and 4CD treatment without lindane did not demonstrate any change in cognitive function over the study period. No change in MDA levels were observed as compared to the control group. However, PREG-S and 4CD treatment group demonstrated a significant increase in brain GSH levels while PROG alone treated group did not show any significant change in the brain GSH levels.4.PROG failed to alter the lindane induced changes in TL, SDL, MDA, GSH. 5.PREG-S or 4CD treatment following lindane administration for 6 weeks was able to reverse the cognitive impairment induced by lindane as shown by improvement in SDL. The two drugs also reversed lindane induced oxidative stress as demonstrated by the decreased MDA and increased GSH levels in these groups compared to lindane only treated groups.6.Lindane induced impairment of TL was not modified PREG-S or 4CD treatment.PREG-S and 4CD were able to reverse the lindane induced cognitive impairment at least in the Step down latency paradigm. The two drugs also reversed the derangement in oxidative stress parameters of MDA and GSH produced by lindane. PROG failed to influence memory impairment and oxidative stress induced by lindane. None of the drugs were able to modulate the changes induced by Lindane in the transfer latency paradigm. Our study reveals a possible correlation between memory impairment and oxidative stress in brain signifying yet another potential role of neurosteroids in the functioning of nervous system and possible use of this group of chemicals in reversing the damage induced by toxicants like lindane in the brain. / Faculty of Medical Sciences, University of Delhi, India

Human African trypanosomiasis (HAT), a lethal disease caused by infection with Trypanosoma brucei rhodesiense or Trypanosoma brucei gambiense, affects a significant number of people in sub-Saharan Africa. Related trypanosome species are also responsible for veterinary trypanosomiasis in many species of domestic and wild animals, causing disruption to agricultural and economic development in one of the world’s poorest areas. Current control and management of the disease mainly relies on a handful of trypanocides that have been in use for over 50 years. The treatment is associated with numerous of limitations such as drug toxicity, affordability and, most worryingly, increasing rates of treatment failure due to spread of parasites resistant to the existing drugs. Therefore, development of new drugs against Human African Trypanosomiasis is much needed. Trypanosomes are incapable of de novo synthesis of the purine rings, and since purines are essential for many cellular functions (e.g. nucleic acid synthesis, energy metabolism) this means the parasites have an absolute requirement for exogenous purines. Several nucleoside and nucleobase transporters have been identified in T. brucei brucei, including the P1 and P2 adenosine. These transporters have recently received a great deal of interest as potential drug targets as possible determinants of drug resistance. The P2 nucleoside transporter is one of the best-studied transporters and it has been shown to facilitate entry of arsenical and diamidine trypanocides, such as melarsoprol and pentamidine. Loss of this transporter appears to confer resistance to some of these drugs in vitro. It has been shown that pentamidine is taken up by two additional transporters named high affinity pentamidine transporter (HAPT1) and low affinity pentamidine transporter (LAPT1). These transporters have been studied based on their kinetic role of drug uptake and neither has been characterised at the molecular level. The main aims of the project were to study the HAPT1 transporter in detail, including identification of substrate recognition determinants, involvement in transport of diamidine trypanocides other than pentamidine, and the cloning of the genes coding for HAPT1 and /or LAPT1 from T. b. brucei. It was found that the main veterinary drug, the diamidine diminazene, was also transported by HAPT1, albeit much less efficiently than pentamidine. This provided new insights into the causes of drug resistance for African trypanosomes, in particular, the well-documented cross-resistance between melaminophenyl arsenical drugs and diamidines. A large number of diamidine analogues and other compounds were tested for inhibition of HAPT1 and their inhibition constants were determined. This identified the essential characteristics for high affinity interaction between substrate and the transporter-binding pocket. This study will aid the design of new ligands and inhibitors for this drug transporter. Furthermore, the gene for HAPT1 was identified as AT-E, a close analogue of TbAT1, which encodes for the P2 transporter. Two distinct alleles, named AT-E1 and AT-E2, were identified in wild-type T. b. brucei, probably representing a single gene locus. Knockdown of AT-E expression using RNAi significantly decreased HAPT1 in both bloodstream forms and procyclics. Expression of AT-E was reduced in the B48 pentamidine resistant line that lacks HAPT-mediated drug uptake. This project has greatly increased our understanding of the biochemical and molecular nature of HAPT1 transporters in T. b. brucei.

Prenatal exposure to maternal infection has been thought as a major risk factor for neurodevelopmental brain damage and thus contributes to the pathophysiology of neurodegenerative diseases including schizophrenia and autism. The mechanisms of aberrant neurodevelopmental processes on the offspring, in which primary cerebral insults occur during early brain development, are not fully understood. In the present investigation, maternal infection was modelled in timed-pregnant rats at embryonic day (E) 14, 16 and 18 by administering intraperitoneal injections of polyriboinosinic-polyribocytidilic acid,poly(I:C), a viral mimetic double stranded RNA complex which activates Toll-Like-Receptor-3 (TLR-3). The aim was to examine the impact of maternal inflammatory response on the regulation of expression of neurodevelopmental proteins that play important roles in many neurodevelopment aspects, including maintenance of synaptic plasticity, intracellular signalling and neurogenesis which may be relevant in cognitive and behavioural functions. An examination of embryo brains 5 h after maternal poly(I:C) showed significant differences in expression of the NMDA receptor NR2 subunits. The expression of NR2A subunits was reduced, whereas infection induced during pregnancy enhanced NR2B subunit expression. Expression levels of both subunits at postnatal day 21 (P21) were not affected by prenatal poly(I:C) exposure. In utero viral challenge led to significant changes among neurogenesis factor only at P21. In the fetal brain, acute poly(I:C) exposure had no effect on the expression of SHH, PCNA and also SOX2 proteins. However, when poly(I:C) was administered during mid and late gestation in the rodent model, long term effects of prenatal viral challenge on survival and maintenance of cell in the brain as indicated by the expression of SOX2 and SHH was clearly demonstrable. Expression of SOX2 level was increased,while SHH was significantly decreased, suggesting possible increase in the number of cells and changes in the rate of differentiation, respectively. The results demonstrate that poly(I:C) challenge in pregnant dams results in selective molecular changes in the brain, with transient alteration in the levels of NMDA receptor subunit NR2A and NR2B in the foetal brain, and also affecting molecules associated with cell genesis processes at later stages of developmental age of offspring. On the other hand, recent pharmacological interest in kynurenines with respect to CNS diseases has mainly focussed on two neuroactive molecules: quinolinic acid (QUIN) and kynurenic acid (KYNA). Manipulation of the kynurenine pathway and its neuroactive metabolites has been associated with N-methyl-D-aspartate (NMDA) receptor neurotoxicity and dysfunction which linked to the development of various neurological disorders. An early developmental event has been proposed to precipitate alterations in the NMDA receptor function. In this respect, early development during the gestational period of rats is most suitable for investigating the modulating effect of kynurenine pathway inhibition by compound Ro61-8048 (3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl]benzenesulphomide) an inhibitor of kynurenine-3-monooxygenase (KMO) in shifting the balance towards the production of neuroprotective, kynurenic acid. Western blots were generated to indicate the expression of a range of proteins relevant to different aspects of CNS development including neuritogenesis, axon guidance, maintenance of synaptic plasticity, intracellular signalling and cell proliferation and migration. Within 5 h of Ro61-8048, there was a significant decrease in NR2A expression and increased NR2B in the embryo brains, with subsequent changes in SHH and NFB at 24 h post treatment. The litters were left undisturbed until weaning on P21 and other groups were allowed to develop to P60, at which time they euthanized and the brains removed for analysis. At P21, western blot analysis revealed significantly increased protein expression of the NR2A and NR2B subunits and postsynaptic density protein (PSD95). Among several neurodevelopmental proteins, the expression of NFB and proliferating cell nuclear antigen (PCNA) was increased, while reduced level of SHH was detected. We demonstrate here persisting changes in NR2A expression, with reduced level in the hippocampus while a raised level was noted in the cortex suggesting prenatal modulation of kynurenine pathway causes long lasting modifications of NMDA receptor composition and function. It is important to note that kynurenine pathway inhibition can generate a consistent set of long term changes in the SHH in which the levels of this protein remained repressed in some regional areas of the brain including hippocampus, cerebellum and cortex. We show that there are some common pathways that are affected by kynurenine pathway inhibition, and this early modulation tends to disrupt critical molecular processes that are known to be actively occurring at each specific developmental time. Overall, given these selective and differing developmental profile, an early life modulation of the kynurenine pathway might be expected to cause a sufficient disturbance of biological processes that are actively occurring at the time of exposure and also able to leave a series of molecular changes that persist into adulthood. This disruption is likely to influence the resulting physiology of the adolescent and adult brain and subsequently can lead to impairments in social behaviour. It is hoped that this study provides a broad analysis of the long term molecular effects of developmental kynurenine metabolism, and that it allows for a viable opportunity of potential therapeutic targets for disease intervention.

The aim of this thesis was to investigate the electrical and mechanical responses to inhibitory non-adrenergic noncholinergic (NANC) nerve stimulation in the bovine retractor penis muscle (BRP) and compare them with those to an inhibitory extract made from this muscle. The extract may contain the NANC inhibitory transmitter of the BRP and possibly of other smooth muscles. Because of species differences in the electrical response to NANC nerves in the rat and rabbit anococcygeus the effects of the extract on these tissues was also investigated. Prior to the investigation of the extract, both the excitatory and inhibitory responses to field stimulation in the BRP, and the effects of passive membrane potential displacement were studied using conventional intra- or extracellular (sucrose gap) recording techniques. The majority of cells in the BRP were electrically quiescent independent of the resting tone. The most frequent (in approximately 25% of preparations) form of spontaneous activity, oscillations in membrane potential and tone, may represent a pacemaker activity. The BRP had cable properties; the time constant and space constant indicated a high membrane resistance. In the absence of tone, field stimulation of the BRP evoked excitatory junction potentials (ejps) in every cell impaled and contractions, graded with the strength, frequency and number of pulses; spikes were not observed. Guanethidine (1-3 x 10-5M) abolished the ejps and contractions, confirming their adrenergic origin. Noradrenaline added exogenously depolarised and contracted the muscle. These effects were blocked by the a-adrenoceptor antagonists, phentolamine and prazosin. However, phentolamine (2.5x 10-6M) inhibited the contraction without reducing the ejp significantly. These effects may be independent of adrenoceptor blockade or the ejp may be mediated by a substance other than noradrenaline (e.g. ATP) released from adrenergic nerves. Prazosin (1.4 x lO-6M) failed to block either the ejp or contraction, indicating the possible existence of two types of adrenoceptor in the BRP; one activated by neuronally-released and the other by exogenously-added noradrenaline. ATP, a contaminant in the extract, also depolarised and contracted the BRP. Physostigmine reduced whilst atropine enhanced the ejps and contractions without similarly affecting the response to exogenous noradrenaline. This confirmed the presence of a cholinergic inhibitory innervation acting on the excitatory adrenergic fibres (Klinge and Sjostrand, 1977). TEA (1 x lO-4M) enhanced the ejp and contraction. Higher concentrations (0.5 to 10 x 10-3M) depolarised, increased the tone and evoked electrical and mechanical oscillations but no spikes. The depolarisation and contraction to exogenous noradrenaline were not enhanced, indicating that TEA acts on the adrenergic nerves. Some post-synaptic effect to block K+ channels also seems likely. The relationship between ejp amplitude and membrane potential in the double sucrose gap was linear and indicated a reversal potential more positive than -30mV. Electrotonic pulse amplitude decreased during the ejp, indicating an increased membrane conductance. Ejps and contractions were reduced following the replacement of the NaCl of the Krebs solution with sodium glutamate. This may be due to the effects of glutamate itself (e.g. Ca2+ chelation) rather than reduction in the membrane Cl- gradient. Tone usually developed spontaneously and was accompanied by membrane depolarisation (from -53 to -45mV) which may open voltage-dependent channels, causing Ca2+ entry and/or its release from intracellular binding sites. Field stimulation produced inhibitory potentials (ijps) and relaxations graded with the strength and number of pulses but showing little frequency dependence. Rebound depolarisation and contraction often followed the ijp and relaxation. Tetrodotoxin (3 x IO-6M), but not adrenergic or cholinergic antagonists, abolished the ijp and relaxation, confirming their non-adrenergic non-cholinergic neurogenic nature. The extract, prepared and acid-activated as described by Gillespie, Hunter and Martin (1981), hyperpolarised and relaxed the BRP, as did sodium nitroprusside and adenosine triphosphate (ATP). Unlike the activated extract or sodium nitroprusside, desensitisation to ATP occurred rapidly and without any change in the inhibitory electrical or mechanical responses to field stimulation. The ijp and relaxation in the BRP were insensitive to apamin but abolished by oxyhaemoglobin (4-8 x 10-6M), as were the responses to extract and sodium nitroprusside. In TEA (10-2M), field stimulation evoked relaxations with no accompanying electrical change. The ijp may be unconnected with or additional to another mechanism producing relaxation. The relationship between membrane potential and ijp in the BRP was non-linear. Ijp amplitude was initially increased during membrane potential displacement from -45mV to approximately -60mV. Thereafter (-60 to -l03mV) the ijp was reduced. Ijps were abolished at -27 and -103mV; reversal was not observed. The hyperpolarisation to extract was also enhanced during passive displacement of the membrane potential to more negative values (-57mV). Membrane resistance increased during the ijp. The extract produced inconsistent changes in membrane resistance, possibly because of the presence of more than one active component. K+ withdrawal failed to enhance the ijp or hyperpolarisation to extract and 20mM K+ did not abolish the the ijp at membrane potentials exceeding EK (-49mV). Thus, the ijp or hyperpolarisation to extract are unlikely to be mediated by an increased K+ conductance. Reducing the Cl- abolished the hyperpolarisation to field stimulation and extract. This occurred more quickly than the anticipated reduction in the Cl- gradient and may be due to Ca2+ chelation by the anion substitute (glutamate or benzenesulphonate) or blockade of the resting conductance which is normally inactivated by the transmitter. Ouabain (1-5x 10-5M), which reduces both the Na+ and Cl- gradients, abolished the ijp, implicating either of these ions as the ionic species involved. In the rat and rabbit anococcygeus, field stimulation and extract each reduced guanethidine-induced tone. This was unaccompanied in the majority of cells in the rat by any significant electrical response. In the remaining cells, inhibition of the membrane potential oscillations occurred. The rabbit anococcygeus differed in that inhibition of the electrical oscillations was observed in every cell exhibiting this behaviour. However, the majority of cells in the rabbit were electrically quiescent and showed only small hyperpolarisations to field stimulation and no electrical response to extract. Apamin (1 x 10-7M) failed to block the electrical and mechanical response to field stimulation in the rabbit but did inhibit transiently that to extract. The latter effect may be due to the initial excitatory effects of apamin. The similarities between the electrical effects of the extract and those of inhibitory nerve stimulation in the BRP, rat and rabbit anococcygeus muscles are generally consistent with their being mediated by the same active component. Moreover, the ijp in the BRP shows properties which have not been reported in other non-adrenergic noncholinergically innervated smooth muscles.

Long-term depression is a long-lasting decrease in signalling between neurons in the central nervous system. It can be elicited via a number of mechanisms, including NMDA receptors and mGLuR receptors. NMDA-dependent LTD is caused by the endocytosis of AMPA receptors in response to stimuli, whereas mGLuR-dependent LTD acts via the action of kinases. Recent studies have demonstrated a novel form of LTD, which is dependent on serine protease action. Molecular changes in protein expression also accompany the generation of this new type of LTD, although the exact mechanism has not been determined. The aims of this current study are to determine the mechanism by which subtilisin could modulate LTD, and whether this effect was solely dependent on proteolytic action, or other factors. Subtilisin-mediated LTD was elicited in hippocampal slices of mice as an experimental model, and each slice was retained at the end of every experiment to enable protein levels to be analysed via immunoblotting. Initial experiments were carried out to evaluate the relationship between subtilisin activity and metalloprotease action, based on previous studies that demonstrated subtilisin proteolysis of the synaptic protein VAMP-1, a known target of the metalloprotease tetanus toxin. The putative link between these two proteases was tested by addition of zinc to the perfusing solution, which promotes the activity of zinc-based metalloproteases. Subtilisin however was completely unaffected by this change to extracellular levels of zinc. Experiments were also conducted in the presence of the metal ion chelators EDTA and captopril to determine the contribution of metal ions to subtilisin action. Addition of the general metal chelator EDTA failed to decrease subtilisin action, and the zinc-specific chelator captopril likewise did not display inhibitory action against subtilisin. Attempts were also made to produce a direct comparison between the action of subtilisin and tetanus toxin, however, preliminary experiments were unsuccessful as the tetanus toxin used in the experiment was unable to cause VAMP-1 proteolysis. The possibility that subtilisin action was solely dependent on their inherent serine protease function was also explored using the general serine protease inhibitor phenylmethanesulfonylfluoride (PMSF). The use of PMSF could provide a better indication of the link between LTD and subtilisin proteolysis, and also shed light on the order of protein degradation. Perfusion of PMSF-inactivated subtilisin prevented the proteolysis of proteins, and also prevented the generation of LTD and abolished the proteolytic activity of subtilisin. These results indicate a close link between the LTD- 2 inducing and proteolytic effects of subtilisin, and in conjunction with the metalloprotease experiments, suggest a serine protease basis for subtilisin-mediated LTD. These results suggested that subtilisin action was not based on metalloprotease degradation of VAMP-1 alone, but on a more general proteolytic effect involving other protein targets such as the netrin receptor Unc5H3 and the cytoskeletal protein actin. The specific role of the Unc5H3 and VAMP-1 proteins in the subtilisin process was studied using protein- specific antibodies to prevent proteolysis. Preincubation of hippocampal slices with VAMP-1 proteins did not prevent the proteolytic effects of subtilisin, nor could it prevent the generation of LTD. Preincubation of hippocampal slices with VAMP-1 antibody in the presence of either the cell permeabiliser triton, or the pore-forming chemical streptolysin also had no inhibitory effect on subtilisin action. Hippocampal slices were also preincubated with Unc5H3 antibodies to determine the importance of this protein to the activity of subtilisin; however, this did not change the proteolytic or electrophysiological effects of subtilisin. Addition of either triton or streptolysin to the preincubation solution containing Unc5H3 antibodies failed to prevent the effects of subtilisin on the hippocampal slice, a result identical to those obtained with VAMP-1 antibody preincubations. The importance of actin proteolysis in mediating subtilisin action was also assessed by addition of the actin stabiliser jasplakinolide to the aCSF used in perfusions to prevent actin degradation. Jasplakinolide was unable to prevent the onset of subtilisin-mediated LTD or its associated degradation of actin. In order to verify whether the effects of subtilisin represented a novel form of LTD, the LTD elicited by subtilisin was compared to that of more established methods of eliciting LTD, such as the mGluR agonist DHPG. Slices exposed to DHPG failed to elicit LTD, however, the GABA mimetic ethylenediamine (EDA) appeared to be a potent inducer of LTD. EDA produced a significantly smaller LTD effect in comparison to subtilisin, which was not accompanied by degradation of any proteins associated with subtilisin perfusion. It would be of interest to know whether other serine proteases could cause this LTD effect, and based on previous studies, it is known that members of the S8A subfamily and the S1A subfamily of serine proteases are capable of causing the same effects as subtilisin. To date much work has been performed on subtilisin, and other groups S8A serine proteases such as Proteinase-K. In comparison, very little is known about of the mechanism utilised by group S1A serine proteases in causing LTD. In order to gain a greater understanding of The possibility that other serine proteases could generate LTD was also investigated as part of this study, with particular emphasis on members of the S8A (to which subtilisin belongs) and S1A subfamily of serine proteases, which are biochemically similar to 3 members of the S8A subfamily. Previous studies have shown that proteinase-K and cadeprin, both members of the S8A, as well as α-chymotrypsin, a member of the S1A subfamily, can replicate this LTD effect. By studying the impact of α-chymotrypsin on fEPSP and protein degradation, it was hoped that this would provide a much more balanced view on the mechanisms behind the LTD-inducing effects of serine proteases. Previous experiments with subtilisin had ruled out the action of mGluRs or the role of electrically-stimulated LTD as the basis for subtilisin-mediated LTD. For this study these two factors were investigated using α-chymotrypsin in order to discover the different mechanisms utilised by these two serine proteases. Electrically-elicited LTD is known to be sensitive to the action of protein phosphatases, and in the light of this knowledge, experiments were conducted using the phosphatase inhibitors phenylarsine oxide (PAO) and sodium orthovanadate. Sodium orthovanadate did not affect the progression of α- chymotrypsin LTD; however, PAO did significantly decrease the magnitude of this type of LTD. This suggests a possible role for tyrosine kinase in this process, although more experiments would be necessary to provide a definite link as sodium orthovanadate had no effect on the action of chymotrypsin. The involvement of mGLuR in α-chymotrypsin LTD was tested using the p38 MAPK inhibitor SB203580. Perfusion of this chemical did not result in a noticeable decrease in α-chymotrypsin activities, and taken together with previous experiments in subtilisin, suggests that serine protease-mediated LTD is not solely dependent on these two pathways for their neuronal depressive actions. The results presented here strongly suggest that LTD caused by serine proteases such as subtilisin and α-chymotrypsin do not involve metalloprotease functions. Instead, LTD is closely linked with the proteolysis of a select number of proteins that are cleaved by the endogenous proteolytic functions of these serine proteases. Furthermore, serine protease- mediated LTD is not simply restricted to subtilisin alone, but can involve other members of the S8A subfamily of serine proteases such as α-chymotrypsin.

Viral respiratory infection exacerbates asthma symptoms in almost all patients with allergic asthma. Asthma symptoms in viral associated asthma exacerbation are often severe and require urgent care as well as hospitalisation. Corticosteroids are the mainstay treatment for asthma. However, they are less effective in treating virus associated asthma exacerbation. The main aim of the thesis is to determine the role of virus infection in airway allergic inflammation and then define the effects of corticosteroids in virus associated exacerbations of airway allergic inflammation. Mice sensitised and challenged with ovalbumin demonstrated most of the main features of asthma including lung cellular inflammation with eosinophilia, early phase asthmatic responses (EAR), late phase asthmatic responses (LAR), and airway hyperresponsiveness (AHR) to methacholine provocations. Treatment with either systemic (dexamethasone: DEX) or inhaled (fluticasone propionate: FP) corticosteroids in the murine ovalbumin allergic airways inflammation model attenuated inflammatory cells influx and eosinophilia, LAR, and the AHR. Influenza A (H1N1/PR8) is the most infective to mice compared to human parainfluenza virus type 3 (HPIV3), and a synthetic dsRNA, poly (I:C). Influenza infection in mice caused a significant increase of inflammatory cell influx in the airways with marked neutrophilia, and AHR. Ovalbumin challenge in the acute course of influenza infection on a murine model of allergic airways inflammation exacerbated the inflammatory cells influx, LAR, and AHR. Treatment with either DEX or FP attenuated the airway cellular inflammation, LAR, but not the AHR. Mice only infected with influenza were resistant to the corticosteroids (DEX and FP) treatment. DEX but not FP showed antiviral activity against HPIV3 and influenza A in vitro. These data suggest that influenza infection in a murine model of allergic airways inflammation exacerbates the inflammation and alters the sensitivity toward corticosteroids. It is also suggested that some elements in the influenza associated exacerbation of murine model of allergic airways inflammation are refractory or not regulated by corticosteroid treatment.

Commercially available multi-ingredient formulas are ingested by the recreationally trained population to optimise training outcomes; however, there remains no convincing evidence in regards to their effectiveness. Thus, the aim of this project was to analyse the effects of three different multi-ingredient supplements on the expected and marketed outcomes. It was hypothesised that the supplements would potentiate the desired body composition, performance and recovery outcomes as claimed. Study 1 was conducted to analyse the effects of combining a 12 weeks resistance training programme with the ingestion of a commercially available carbohydrate-protein-creatine based multi-ingredient supplement on strength performance and body omposition in recreationally trained males. It was hypothesised that the ingestion the multi-ingredient supplement would potentiate strength performance adaptations to a greater extent than a maltodextrin placebo. As a secondary hypothesis it was expected that ingesting the multi-ingredient supplement would benefit body composition outcomes in comparison to the placebo. Thirteen healthy male subjects were assigned to either a multi-ingredient formula (n=7) or a carbohydrate placebo (n=6). Both groups ingested the multi-ingredient supplement or placebo in the morning and immediately after training. Before and after the 12 weeks progressive resistance training; percentage body fat and fat free mass were determined. Maximum strength and repetitions to failure with 60% one repetition maximum on bench press and parallel squat were also assessed before and after the resistance training period. No significant increases in any of the performance or body composition variables were observed in either group. However, larger standardised effects sizes and magnitude-based inferences demonstrated that the addition of the multi-ingredient supplement to a 12 week progressive resistance training protocol could be effective to potentiate upper body maximum strength or muscular endurance performance, but not body composition outcomes. Study 2 was undertaken in order to analyse the acute effects of a commercially available carbohydrate and caffeine gel on intermittent sprint performance in twelve recreationally trained males. It was hypothesised that the combination of carbohydrate and caffeine in gel form would attenuate fatigue and decrease perception of effort when compared to the ingestion of carbohydrate gels alone and placebo gels. A secondary hypothesis postulated that the carbohydrate and caffeine gel would maintain blood glucose levels throughout the intermittent sprint test in regards to both the carbohydrate and placebo gels. Using a cross-over design, one 70 mL dose of gel containing either, 25 g of carbohydrate with or without 100 mg of caffeine or a non-caloric placebo was ingested on three occasions: one hour before, immediately prior and during the intermittent repeated sprint test. Blood glucose, rating of perceived exertion and fatigue index were analysed. The main finding was that ingesting the carbohydrate and caffeine gel one hour before, prior to and during an IST is effective at transiently reducing fatigue and RPE whilst maintaining higher glucose levels at the final stages of the exercise. Study 3 was conducted in order to analyse the acute effects of a commercially available carbohydrate and protein based multi-ingredient recovery formula on the recovery process and muscle damage, in 10 recreationally trained males, after performing a bout of intermittent sprint exercise. It was hypothesised that the ingestion of a carbohydrate and protein based multi-ingredient supplement, before, during and after an acute bout of intermittent repeated sprint exercise would promote recovery estimated through the attenuation of neuromuscular fatigue and markers of muscle damage respect to the ingestion of carbohydrate only or a low caloric placebo. As a secondary hypothesis the ingestion of the multi nutrient formula would attenuate a decline in sprint performance during the intermittent sprint test when compared to the carbohydrate and placebo conditions. Using a cross-over design, one 500 mL dose of a multi-ingredient recovery beverage, a carbohydrate beverage or a placebo beverage was divided in to 4 equal servings of 125 mL and ingested before each of the 4 blocks of the intermittent sprint test. A second full serving was ingested with 20 minutes of completing the intermittent sprint test. 15m sprint times, creatine kinase, myoglobin, and interleukin-6 were assessed before (pre), immediately post (post), 1 hour and 24 hour after exercise. Total sprint time measured during the intermittent protocol was not different between conditions. 15m sprint time was slower at post, 1 hour and 24 hour compared to pre without differences between conditions. Creatine kinase at 24 hour was lower in the multi-ingredient compared to both carbohydrate and placebo. Myoglobin increased in all three conditions at post, and 1 hour compared to pre, showing lower values at 1 hour for the carbohydrate and approached significance (p=0.060) for multi-ingredient compared to placebo condition. Interleukin-6 increased at both post and 1 hour compared to pre with no differences between conditions. Thus demonstrating, the ingestion of a multi-ingredient supplement before, during and immediately after a 90 min intermittent sprint test resulted in no effects on performance and fatigue. However, the accumulation of some biomarkers of muscle damage could be attenuated. It would appear that manufacturers make use of the research available to formulate multi-ingredient supplements as the supplements that showed efficacy in studies 1 and 2 possess the most established research. Whereas investigations and knowledge in to the acute effects of a carbohydrate-protein based multi-ingredient supplement on recovery from an intermittent sprint test (study 3) still need to be determined.

This thesis reports an ethnomethodological, conversation analytic study of communication between stroke patients and physiotherapists. The study sought to describe and explicate patterns of conduct by which therapists and patients communicate about treatment activities during therapy sessions. Analysis included a comparison between practices observed in the data and current published recommendations for good practice. The data consist of 74 treatment sessions that were video-recorded in four English hospitals. The 21 patient participants were undergoing inpatient rehabilitation for stroke. Most were recorded on four occasions over a two-week period. Their disabilities varied, but all could speak and understand at least short sentences in English. Each of the ten therapist participants was employed at senior level and used treatment approaches that are prevalent in the UK. Analysis involved repeated viewing of data and transcription of talk and body movement. It focused on three areas that emerged as central to physiotherapy interactions: The nature of treatment activities and of participation in them Achievement (success and failure) in these activities Reasons, goals and purposes underlying them Consistent with conversation analytic studies in other settings, we found that each communication practice in physiotherapy has a range of interactional effects, and that these are locally constructed and accomplished. Therefore, rather than generating „blanket prescriptions‟ about „good‟ and „bad‟ interactional practices, our study contributes to enhancing practitioners‟ understanding of the contingencies and underlying orientations that shape communication conduct, and raising their awareness of the effects of different means of achieving various interactional tasks in physiotherapy. We argue that these understandings can contribute to improvements in the practice and training of physiotherapy communication. Our study contributes to ethnomethodological and conversation analytic knowledge regarding methodological strategies for researching lay professional interactions, and to sociological understandings about the organisation of conduct in clinical interactions, particularly the role of orientations to managing physical incompetence and its implications.

Cytotoxic T lymphocytes are able to identify malignant cells by scanning for aberrant peptides presented on cell surface human leukocyte antigen (HLA) Class I molecules by virtue of an antigen binding receptor called the T-cell receptor (TCR). Peptides presented by HLA Class I complexes represent the largest array of tumour associated antigens (TAAs) and are therefore ideal targets for immunotherapeutic reagents. Cancer patients frequently mount T-cell responses to tumour-specific antigens, but these are in most cases ineffective at clearing the tumour. This is in part due to the low affinity of TCRs for self-antigens coupled with low-level expression of target peptides on the surface of cancer cells. To harness the exquisite antigen recognition property of TCRs for use as potential therapeutic proteins, the principal goal of this thesis was to generate ultra-high affinity TCRs against three clinically relevant HLA Class I melanoma-specific epitopes, including peptides derived from Melan-A/MART-1(26-35), gp100(280-288) and MAGE-A3(168-176). TCRs are membrane-bound disulphide (ds)-linked heterodimers consisting of an alpha and a beta chain. Each chain comprises three hypervariable or complementarity-determining region (CDR) loops, which assemble to form the antigen binding domains. As a general rule the CDR3 loops, and to a lesser extent the CDR1 loops, contact the peptide bound in the HLA groove and as such specificity is largely attributable to the CDR3 loops. The remaining CDR loops interact with the HLA surface and not the bound peptide. Each CDR loop was mutagenised using degenerative NNK oligonucleotides and expressed on the surface of bacteriophage as fusions to the phage coat protein pIII. Through a Darwinian process of in vitro evolution using pHLA ligand as the target molecule, mutated TCRs with improved affinity for pHLA were identified. TCRs engineered by phage display were produced as soluble ds-linked proteins and the contribution to affinity of each mutated CDR was measured by surface plasmon resonance (SPR). Using a combinatorial strategy, individual mutated CDRs were spliced into the same TCR molecule in a stepwise manner to further increase binding affinity. The final combination of mutated CDRs was shown to bind their cognate pHLA antigen with substantially improved KD values of 18 pM (Melan-A/MART-1(26-35)), 11 pM (gp100(280-288)) and 58 pM (MAGE-A3(168-176)), representing an increase over the wild-type TCR of approximately 1.8 million-fold, 1.7 million-fold and 3.7 million-fold respectively. In addition, having discovered an off-target binding profile for the high affinity MAGE-A3 TCR, the phage display methodologies were explored to 5 reestablish the specificity of this molecule. These results are significant because this has provided a platform on which, for the first time, to make TCR-based therapeutics. For example, the affinity enhanced gp100 TCR is currently undergoing clinical evaluation in a Phase I/II trial.

Interparticulate interactions play a significant role in determining the downstream behaviours of all pharmaceutical formulations and are therefore essential considerations when approaching formulation design. Inhalation product formulation in particular is inherently bound to an understanding of these forces. Delivery of drugs to the lower airways to treat conditions like asthma and COPD requires a particle size of below 5 micron. This implicitly demands micronization of the active pharmaceutical ingredients (APls) and this process renders many particles of large surface area with high surface energies and an auto-adhesive tendency. There is therefore a concurrent reduction in the flowability and dispersion properties of these systems. The interactive character predisposes agglomeration, flocculation or device retention and will compromise manufacture, stability, device function, and the aerosolization behavior of a formulation. Ultimately the ability of any aerosolized API to reach the deep airways is dependent upon adhesion force dynamics. As such, an appreciation of the forces of attraction and scale of particulate interactions within inhaler technology is critical if a successful drug delivery device is to be realized. The advancement of the atomic force microscope (AFM) as a force probing apparatus, has meant that it is now possible to measure the force of adhesion between two particles of interest. However these measurements could not easily be compared, because there is no simple means to account for differences in the contact regime (geometrics) between measurements. However, the development of the cohesive adhesive balance (CAB) approach by Begat, Morton, Stainforth and Price in 2004 has offered a means to negate this limitation. Using a colloidal probe microscopy (CPM) derived technique a particle of a selected material of interest (API, carrier molecule etc.) is attached to an AFM cantilever and ramped onto and off the surface of another material of interest (adhesion measurement), and to a surface of the same material as the tip (cohesion measurement). By graphically plotting the adhesive force values of a series of tips, as a function of the cohesive force values of the same tips, a representation of the relative particle interaction can be obtained. Quantitative information regarding the adhesive/cohesive nature of the interaction can then be extracted from the graph and a description of the interaction formulated that can be compared to other material combinations. The CAB work carried out to date has used recrystallized model substrates. These molecularly flat surfaces ensured there would be no difference between the contact geometry of a functionalised AFM probe and the adhesive and cohesive surfaces of the study respectively. In this fashion the only variable between the two measurements would be the chemical interactivity, and not the interactive surface area. However while using such methodology guarantees the validity of the approach, it is not necessarily a true representation of the materials 'in-situ' and requires more complex sample preparation and complex experimental design. For a variety of reasons this can be misleading in its own right. This thesis details the .investigation into the application of an adapted CAB approach in characterizing the force balance between APls for inhalation in their real state. In so doing, the aim was to see whether such a CAB would offer a quicker and simpler, yet relevant and informative assessment of a drug system force balance. It was hoped that said force balance could in turn be associated with a measurable impact upon the formulation performance of the characterised ingredients as measured 'in-vitro'. This interest was particularly directed at the lesser characterized pressurized metered dose inhaler (pMOI) systems. While these formulations are solvent based, it was of interest to identify whether a simple API to API challenge could infer a descriptive balance that could link to 'in-vitro' performance. Furthermore there was interest in evaluating the use of a range of surface specific imaging techniques to analyse the deposition dynamics of the combination formulations. It was hoped that by doing so, the localisation of the individual components within the binary deposits could again be associated back to the force balance of that system, and that an appreciation of the capability of the techniques involved would be gained. The work that follows therefore commences with the evaluation and description of the capacity for the CAB approach to be adapted to measure force relationships between real beclomethasone dipropionate (BOP) particles and pMDI component surfaces. From this assessment it was found that even with relatively smooth substrates, the combination of bulky functional particles and the inherent substrate roughness caused a critical failure in the CAB model. The parity between cohesive and adhesive geometries of contact was excessively stretched, leading to a loss of force normalisation which was reflected in uncorrelated CAB plots. As a consequence little could be confidently gleaned from the force data acquired, although there was the suggestion that the use of a fluorinated ethylene proplylene (FEP) coating reduced the adhesive interaction between the APls and the pMDI canister wall. This was then followed by an attempt to find a compromise between the model crystal substrates of a pure CAB process and the real particle morphologies that had caused the CAB model to fail. Using a compression process to form API powder compacts, in conjunction with small and discreet functional particles, a confident CAB was achieved for two combinations of APls selected on the basis of surface energy and physical stability analysis. Salbutamol sulphate was characterised with beclomethasone dipropionate, and salmeterol xinafoate with fluticasone propionate. Both combinations showed CAB plots with a sufficiently strong linear regression analysis to suggest a broad accuracy of force balance assessment. Both beta2-agonists showed cohesively dominated relationships with respect to the paired glucocortiocoids, while in reverse both glucocorticoids showed adhesively dominated relationships with the beta2-agonists. There was concern raised over the compression process of the powder discs, and its impact on the physicochemical state of the APls, with some thermodynamic evidence of polymorphic changes that required further work. The next chapter looks at the 'in-vitro' deposition performance of the two API combinations from a HFA134a pMDI system by analysis in an Andersen Cascade Impactor (ACI). The coformulation of salmeterol with fluticasone induced an improvement in the fine particle performance of fluticasone, with a concurrent decrease in the fine particle performance of salmeterol. This impact was hypothesised to be related to alterations in the structure and strength of particle-particle agglomerates. The impact on deposition performance of coformulating beclomethasone and salbutamol was unclear, as a critical unexplained loss of beclomethasone by total recovered mass was seen from all beclomethasone containing formulations. This instability of beclomethasone within the HFA134a system, precluded an accurate assessment of a direct impact on salbutamol deposition. The final chapter, compared a range of surface specific imaging techniques, including scanning electron microscopy (SEM), desorption electrospray ionization mass spectrometry (DESI), Raman spectrometry and time-of-flight secondary ion mass spectrometry (ToF-SIMS) in assessing the extent and nature of 'in-vitro' co-deposition from the salmeterol and fluticasone pMDI formulations. It was apparent that the deposition of the two APls on ACI plates was not likely to be directly comparable assessment of the incidence of particle co-deposition 'in-vivo' due to the forced nature of nozzle directed impaction. However the combination of techniques employed produced a wealth of physical and chemical data that did suggest that the two APls showed extensive co-ordination 'in-vitro'. Raman spectroscopy was able to identify individual particle character and showed frequent salmeterol and fluticasone particle combinations, but suffered from exceptionally long run times and anomalies from photoreactive surface elements. The use of a multivariate approach to ToF-SIMs analysis defined the strong co-association of the two APls, although could not differentiate particle to particle deposition. Multivariate curve resolution (MeR) was used and produced distinct components that segregated ions from both APIS from the background plate but not from each other. SEM imaging was able to define the morphologies of the deposited particles, but was unable to differentiate the two. DES I imaging showed the presence of the two APls together within several drug spots, but could not be used to investigate individual drug spots, and the distribution within, due to inadequate spatial resolution and differences in desorption efficacy. While the co-association of the two APls was observed, the lack of a comparator in another combination of APls made the link between deposition performance and force balance purely descriptive. It was unclear as to whether the force balance of the system lends itself to a particular increase in co-deposition behaviour. However it was apparent that the analytical techniques employed all had respective strengths and weaknesses as mapping tools, and with further work with other formulations could be used to provide a tailored formulation screening process, if subsequent links to force balances could be made.