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Background: A number of systematic reviews provide the basis for decision-making in treatment planning of the reconstruction of mutilated dentitions. This includes identifying teeth difficult to treat/save and decisions on replacement such teeth if extracted. Aims: 1) To assess attitudes of general dental practitioners (GDPs) in a community where provision of dental implants is a widely practiced treatment; and to determine variations in attitudes with respect to dentists’ factors, training factors and implant-provision factors (Study 1): and 2) To determine treatment decision making with respect to maintaining periodontally compromised teeth among dentists with and without postgraduate qualifications in implant dentistry (Study 2). Methods: Study 1:A cross-sectional survey was conducted on a random sample of registered dentists in Hong Kong regarding their attitudes towards implant dentistry with respect to (1) perceived superiority, (2) perceived outcomes, (3) perceived complications and maintenance issues and (4) placement issues. In addition, information was collected on dentists’ factors, training factors and implant provision factors. Variations in attitudes towards implant dentistry were explored. Study 2: A series ofpatient scenarios with varying degrees of periodontal disease levels was presented to selected dentists. Information on their decision-making outcomes, and their intention to retain compromised teeth was analysed in bivariate and regression analyses; accounting for postgraduate implant training, gender, years in dental practice and implant placement experience. Results: Study 1: Among eligible practitioners (n=246), the response rate was 46.3%. Most dentists’perceived implants to be superior to conventional prostheses for the replacement of a single missing posterior tooth (80%) and likewise, for the replacement of a single missing anterior tooth (67%). Variations in attitudes existed with respect to dentists’ factors: years in practice (p<0.05), place of graduation (p<0.05); implant training factors (p<0.05), number of days of training (p<0.05) and implant experience factors (p<0.05). Study 2:This study involved 30 dentists with postgraduate implant qualifications (GDPP), 33 dentists without postgraduate implant qualifications (GDP) and 27 dentists undergoing postgraduate training for implant qualifications (GDPT). Variations in treatment decision-making were evident between the three groups (p<0.05). Differences in treatment approaches to retaining compromised teeth were apparent(p<0.05). Furthermore, variations in rehabilitation of extracted scenarios existed in terms of use of implants and number of implants needed for rehabilitation. Accounting for dentist and practice factors in regression analyses, compared to GDP, GDPP/GDPT were three times as likely to retain periodontally compromised upper molars with painwith pain (OR 3.08, 95%CI 1.09, 8.14 p=0.03), or without pain (OR 3.10, 95%CI 1.04, 10.62 p=0.04). Conclusions: Study 1:In a community where provision of dental implants is widespread the attitudes of GDPs are not wholly in line with evidence-based knowledge. Variations in their attitudes exist with respect to dentist factors, training and experience issues. Study 2: Variations in treatment decision making with respect to retaining periodontally compromised teeth exist between dentists with and dentists without postgraduate training in implant dentistry. Furthermore, there are differences in their management approaches. / published_or_final_version / Dentistry / Master / Master of Philosophy

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.