Recent studies suggest that the neural retinal response to light is compromised in diabetes. Electroretinogram studies suggest that the dim light retinal rod pathway is especially susceptible to diabetic damage. The purpose of this study was to determine whether diabetes alters rod pathway signaling.

Le canal sodique épithélial sensible à l'amiloride (ENaC) est une protéine composée de trois sous unités similaires [alpha][bêta][gamma] codées par trois gènes distincts et associées en un complexe hétérotétramérique [alpha]2[bêta][gamma]. ENaC est localisée au niveau de la membrane apicale des cellules épithéliales à jonctions serrées. C'est une protéine clé de la réabsorption du sodium au niveau du néphron distal, des voies aériennes supérieures et du colon distal. Son rôle dans la régulation du volume extracellulaire et la pression sanguine a été renforcé par la découverte de deux maladies génétiques humaines liées à la fonction du canal : le syndrome de Liddle et le pseudoaldostéronisme de type I (PHA I) due respectivement à un gain et une perte de fonction du canal. L'activité et l'expression de ce dernier sont finement régulées. Sa régulation par les sérines protéases et le sodium extracellulaire (auto-inhibition) a été mis en évidence par Chraïbi et collaborateurs en 1997, mais peu de choses sont connues sur les mécanismes et le rôle fonctionnel de la boucle extracellulaire dans ces différentes voies de régulation. Notre objectif est d'identifier les sites potentiels impliqués dans ces différentes voies de régulation. Pour ce faire, des mutations au niveau de la partie terminale de la boucle extracellulaire des sous unités [alpha], [bêta] et [gamma] du canal ont été réalisées par mutagenèse dirigée et l'effet des sérines protéases et du sodium extracellulaire a été étudié. Nos résultats, obtenus par la technique de voltage-clamp à deux électrodes, montrent que le domaine WPS (un domaine conservé dans les trois sous unités des différentes espèces et situé au niveau de la partie terminale de la boucle extracellulaire du canal) est impliqué dans la régulation d'ENaC par les sérines protéases et le sodium extracellulaire. Nous avons montré plus particulièrement que le tryptophane [alpha]W453 a un rôle fonctionnel majeur dans ces deux voies de régulation. Sa substitution par une arginine au niveau de la sous unité [alpha] rend ENaC résistant à la trypsine et abolit totalement l'inactivation du canal par le sodium extracellulaire. Par contre, la même mutation au niveau de la sous unité [gamma] ne change ni la réponse du canal à la trypsine ni sa sensibilité au sodium extracellulaire, alors que les canaux portant la mutation W/R au niveau de la sous unité [bêta] ont une sensibilité intermédiaire. L'ensemble de ces résultats montre le rôle fonctionnel de la boucle extracellulaire d'ENaC dans la régulation du canal par les sérine-protéases et le sodium extracellulaire.

Nitric oxide is released from the endothelium and causes relaxation of vascular smooth muscle by stimulating guanylate cyclase to produce guanosine 3’,5’-cyclic monophosphate (cGMP) which, in turn, is degraded by phosphodiesterase type 5 (PDE5). Inhibition of PDE5, with drugs like sildenafil citrate, promotes NOstimulated relaxation of vascular smooth muscle. The overall aim of the work contained within this thesis was to further characterise the systemic vascular effects of PDE5 inhibition. Four clinical studies were performed. The aims of the first study were to investigate in healthy men the effect of smoking on endothelium-dependent vasomotor function measured as the change in peripheral arterial wave reflection with inhaled salbutamol, and the effect of acute sildenafil 100 mg on this response. Smokers (n=12) exhibited a reduced response to inhaled salbutamol compared to non-smokers (n=11) [mean(standard deviation) area under the curve of the change in central augmentation index following salbutamol 400 μg: -29(143) AU in smokers vs -159(124) AU in non-smokers, P=0.03]. In the smokers, there was a trend to an improvement in the response to salbutamol following sildenafil [-96(266) AU vs -29(143) AU with matched placebo; P=0.2]. The co-administration of glyceryl trinitrate (GTN) and sildenafil is absolutely contraindicated because of the potential for profound hypotension. The aim of the second study was to characterise the time course of this interaction. Twenty men with stable angina, maintained on their usual medicines, were administered sublingual GTN 400 μg 1, 4, 6 and 8 hours after sildenafil 100 mg or matched placebo. Compared to the combination of GTN and placebo, the combination of GTN and sildenafil resulted in greater mean maximum reductions from baseline in sitting systolic blood pressure (BP) at 1, 4 and 8 hours, and in sitting diastolic BP at all time points (all P<0.05). Compared to placebo, sildenafil alone reduced systolic BP at 1, 4, 6 and 8 hours (P<0.01 at 1 hour and P<0.05 at 4, 6, and 8 hours) and diastolic BP at 4, 6, and 8 hours (all P<0.01). Analysis of the change in BP from the measures taken before each GTN challenge suggested that the interaction on BP might be synergistic at 1 hour after sildenafil, but no more than additive at 6 and 8 hours after sildenafil. Symptoms consistent with hypotension occurred following GTN in 6 subjects at 1 hour and 3 subjects at 4 hours after sildenafil, but in no subjects at 6 and 8 hours after sildenafil or at any time after placebo. In the third study, 25 otherwise untreated hypertensives were given sildenafil 50 mg or matched placebo three times daily for 16 days and the effects on ambulatory BP, clinic BP, arterial wave reflection, carotid-femoral pulse wave velocity and brachial artery flow-mediated dilatation were measured. Three subjects were withdrawn because of side effects and the data from the remaining 22 subjects were analysed. Sildenafil reduced ambulatory BP [change from baseline in average daytime BP: systolic -8(9) mmHg vs 2(9) mmHg with placebo, P<0.01; diastolic -6(5) mmHg vs 0(6) mmHg, P<0.01] and clinic BP [change from baseline to 1 hour after drug administration on day 16: systolic -5(11) mmHg vs 4(10) mmHg, P<0.01; diastolic -5(5) mmHg vs 2(7) mmHg, P<0.01]. Sildenafil, but not placebo, reduced arterial wave reflection [central augmentation index from 32(9)% at baseline to 30(10)% at 1 hour after administration on day 16, P<0.05; radial augmentation index from 88(13)% to 84(13)%, P<0.01], but the change in arterial wave reflection was not statistically significant compared to the change with placebo. Sildenafil did not affect pulse wave velocity or flow-mediated dilatation. The fourth study investigated the potential of combined PDE5 inhibition and organic nitrate for the management of treatment-resistant hypertension (TRH). In 6 patients with TRH, maintained on their usual antihypertensives sildenafil 50 mg alone, isosorbide mononitrate (ISMN) 10 mg alone and co-administered sildenafil and ISMN all acutely reduced systolic BP and diastolic BP compared to placebo (quantified as the area under the curve of the change from baseline to 4 hours after drug administration; all P≤0.01). The combination produced a greater reduction in systolic BP than did either sildenafil alone (P=0.03) or ISMN alone (P=0.01) and a greater reduction in diastolic BP than did sildenafil alone (P=0.02). Compared to placebo, from 1 to 3 hours after drug administration BP was on average 13/10 mmHg lower with sildenafil alone, 18/14 mmHg lower with ISMN alone and 26/18 mmHg lower with the combination. The following conclusions were made. (1) Smokers exhibit impaired vascular responsiveness to inhaled salbutamol, indicating systemic endothelial dysfunction, which may be improved by sildenafil. (2) In men with stable angina there is an interaction on BP reduction between sildenafil 100 mg and sublingual GTN 400 μg for at least 8 hours after sildenafil administration, but this interaction is no more than additive from 6 hours after sildenafil administration. (3) Regular sildenafil monotherapy reduces BP in hypertension. (4) In patients with TRH maintained on their usual antihypertensives sildenafil alone and ISMN alone both acutely reduce BP and there is additional BP reduction when these drugs are given in combination.

This thesis takes a lifespan approach to investigate attentional bias from childhood into older adulthood. Using the dot-probe task throughout, the primary aim was to identify age-related differences in attentional bias across the lifespan. Short and longer stimulus presentation times were used in some studies to investigate the time course of attentional bias. Furthermore, anxiety and executive function were measured to examine how these factors may influence attentional bias across the lifespan. Results found that children showed an attentional bias away from emotion faces which was most evident in those with low trait anxiety. Young adults attended to angry faces at the short presentation time, this was not maintained at longer presentation times. In older adults, results showed an initial avoidance of happy faces followed by a bias towards happy faces at the later presentation time. A direct comparison between children and young adults found that children showed avoidance of emotion compared to adults. A direct comparison of young and older adults found in those with higher state anxiety, young adults showed a bias towards threat at the long presentation time, whereas older adults showed a bias away from threat. Contrary to the predominant theory of attention, executive function was not found to be related to attentional bias in children or young adults. However it did influence attentional bias in older adults, where poorer inhibition was related to a bias away from the happy face. To summarise, this thesis has identified differences in attentional bias according to age and prompts further research into how age, anxiety, executive function and attentional bias may interact in a non-clinically anxious population.

Anaerobic ammonium oxidizing (Anammox) bacteria are known to utilize ammonium and nitrite as electron donor and acceptor, respectively, to produce nitrogen gas as their main final product with by-product formation of nitrate. Anammox bacteria provide the advantages of significant saving in aeration, no requirement for external electron donor, reduction of greenhouse gas emission, lowered sludge production, and higher specific nitrogen-removing activity compared to the conventional nitrification-denitrification process used in nutritent-N removal. Therefore, the anammox process has recently been widely studied and applied as a state-of-the-art biotechnology to remove nutrient nitrogen from ammonium-rich wastewater. However, the inhibitory impact of nitrite (one of the two main substrates) on the anammox process has been reported in both lab- and full-scale anammox systems, which limits the application of anammox process. Based on the current knowledge, a wide range of nitrite concentrations causing anammmox inhibition was reported to be correlated to the pH and energy status of anammox bacteria, and the understanding of the mechanisms of nitrite inhibition to anammox bacteria is still not clear. Therefore, the purpose of this work is to investigate the mechanism of nitrite inhibition and develop a strategy for recovering nitrite inhibited anammox processes. The effects of pre-exposing anammox bacteria to nitrite alone on their subsequent activity and metabolism after ammonium has been added was evaluated in batch bioassays. The results showed that pre-exposure of anammox bacteria to nitrite without ammonium caused dramatic inhibition with observed 50% inhibition concentration (IC₅₀) of 52 mg NO₂⁻-N L⁻¹, compared to an IC₅₀ of 384 mg NO₂⁻-N L⁻¹ obtained in the control group with ammonium and nitrite added simultaneously. The accumulated nitric oxide (NO) found in the group with anammox bacteria pre-inhibited by nitrite indicated that pre-exposure to nitrite most likely caused disruption of the anammox biochemistry by interrupting the hydrazine synthesis step. Meanwhile, active metabolic status of anammox bacteria fueled by a strong proton gradient maintained by controlling pH in the optimal range of 7.2-7.8 enhanced the ability of anammox bacteria to tolerate nitrite inhibition. This was evaluated by depleting the proton gradient by utilizing two uncouplers of respiration, 2,4 dinitrophenol (24DNP) and carbonyl cyanide m-chlorophenyl hydrazine (CCCP). The results showed that presence of 0.28 mg CCCP L⁻¹ caused enhancement of nitrite inhibition to anammox bacteria, with a calculated IC₅₀ of 18.7 mg NO₂⁻-N L⁻¹ compared to an IC₅₀ greater than 150 mg NO₂⁻-N L⁻¹ in the control group lacking CCCP. Meanwhile, the sensitivity to NO₂⁻ was 3 times in anammox bacteria pre-exposed to 100 mg NO₂⁻ L⁻¹ for 24 h than in treatments lacking 37.8 mg 24DNP L⁻¹. A potential strategy of detoxifying the nitrite inhibition to anammox bacteria was proposed by using nitrate due to the finding of the presence of NarK, with potential function of NO₃⁻/NO₂⁻ antiporter, encoded in the anammox genome. Both batch- and continuous-experiments were carried out to test this hypothesis. The relative contribution of nitrate to nitrite detoxification was found to be pH dependent but the attenuation of nitrite inhibition is independent of the proton motive force which is supported by the result that nitrate caused almost complete attenuation of nitrite toxicity in cells exposed to the proton gradient disruptor, CCCP, at pH 7.5. Increase in nitrate concentration also improved the attenuation of nitrite inhibition to anammox process, with the maximum recovery being achieved at 0.85 mM in batch experiment and 2.0 mM for 3 days in continuous-fed bioreactor. Moreover, the timing of nitrate addition is significant because long-term nitrite inhibition of anammox biomass results in irreversible damage of the cells, under which condition addition of nitrate showed no positive impact on recovery of nitrite inhibition. This study also investigated the inhibitory effects of six metals (Cu²⁺, Cd²⁺, Ni²⁺, Zn²⁺, Pb²⁺, and molybdate) commonly found in landfill leachate on anammox activity. Results from batch bioassays indicated that precipitation reactions decreased considerably the soluble concentration of the cationic metals. Cu, Zn, Cd, and Ni were the most toxic metals with 50% inhibiting soluble concentrations of 4.2, 7.6, 11.2, and 48.6 mg L⁻¹, respectively. Molybdate and Pb²⁺ were not or only moderately inhibitory at the highest soluble concentrations tested (22.7 mg Mo L-1 and 6.0 mg Pb L⁻¹, respectively). Microbial inhibition was strongly correlated with both the added- and the dissolved metal concentration. These relationships could be described by a noncompetitive inhibition model for all inhibitory metals except for Pb. The results of this dissertation indicate that the resistance of anammox bacteria to nitrite inhibition could be enhanced by maintaining either an active metabolism in simultaneous presence of ammonium and nitrite, or sufficient proton gradient to enable relieving nitrite accumulation in sensitive regions of the anammox cells through an active nitrite transport system. An alternative nitrite detoxification mechanism was also demonstrated which relied on a secondary transport system facilitated by exogenous nitrate to avoid the accumulation of toxic intraorganelle nitrite concentration. Moreover, the results obtained in the study investigating the impact of heavy metals on anammox process provides new insights on the sensitivity of anammox bacteria to common metals and can be used to devise strategies to minimize inhibition of the anammox process when treating wastewater containing heavy metals.

We assessed age differences in the ability to resolve competition for figural status in stationary displays using small, enclosed, symmetrical silhouettes that participants classified as depicting "novel'' or "familiar'' shapes. The silhouettes were biased such that the inside was perceived as the shaped figure, and the outside was perceived as a shapeless ground. The critical manipulation was whether a portion of a meaningful object was suggested on the outside of the border of some of the novel silhouettes but not others M(+)Ground and M-Ground novel silhouettes, respectively). This manipulation was intended to induce greater inhibitory competition for figural status from the groundside in M(+)Ground silhouettes than M(-)Ground silhouettes. In previous studies, young adults classified M(+)Ground silhouettes as "novel'' faster than M(-)Ground silhouettes (Trujillo, Allen, Schnyer, & Peterson, 2010), suggesting that young adults may recruit more inhibition to resolve figure-ground when there is more competition. We replicated this effect with young adults in the present study, but older adults showed the opposite pattern and were less accurate in classifying M(+)Ground than M(-)Ground silhouettes. These results extend the evidence for inhibitory deficits in older adults to figure assignment in stationary displays. The (M(+)Ground - M(-)Ground) RT differences were evident in observers' longest responses, consistent with the hypothesis that inhibitory deficits are evident when the need for inhibition is substantial.

This thesis examines the design, synthesis and biological applications of a series of inhibitors which incorporate an azobenzene photoswitch, a peptidyl backbone and a trifluoromethyl ketone warhead. The photoswitch can be isomerised by irradiation with UV or visible light and has been employed to modulate the reactivity of the enzyme. Chapter one gives a brief outline of some of the important areas related to this work. Examples of previously utilised photoswitches as well as some background on serine protease and the uses of fluorine in medicine has been covered. Chapter two outlines the synthesis of the key trifluoromethyl carbinol 2.6 by two different methods. The condensation of a fluorinated aldehyde with a nitroalkane affords an α-nitro trifluoromethyl carbinol which can be reduced to give the desired amine 2.6. Treatment of oxazolones with trifluoroacetic anhydride via a modified Dakin-West reaction gives trifluoromethyl ketones which can be reduced to give trifluoromethyl carbinols. Chapter three investigate the synthesis of substituted stilbenes and phenanthrenes as alternative molecular switches to azobenzenes. Molecular modelling of phenanthrenes suggests they may be suitable mimics of E-azobenzenes. Chapter four outlines the synthesis of a series of mono and disubstituted azobenzenes by direct sulfonation of azobenzene or by condensation of nitroso arenes with aryl amines. The switches incorporate one or two peptidyl residues designed to improve specificity towards the enzyme. Chapter five examines the photoisomerisation of eight potential inhibitors by irradiating with UV or visible light. Irradiation with UV light enriches the sample to give 78-93 % of the Z-isomer. Irradiation with visible light gave photostationary states with 14-21 % Z-isomer. Ambient photostationary states are ca. 22 % Z-isomer. Chapter six looks at the testing of five trifluoromethyl ketones as potential inhibitors ofα-chymotrypsin. The inhibitors vary in substituents, substitution patterns and chain length. The inhibitors were tested at both ambient and Z-enriched photostationary states and were found to exhibit slow binding kinetics. In all cases the Z-enriched inhibitor solution was at least 3-fold more potent than the ambient solution. Chapter seven is an experimental chapter and outlines the synthesis of the compounds prepared in this thesis.

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