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Latrophilin1 (LPHN1) is a presynaptic adhesion G protein-coupled receptor involved in the control of spontaneous exocytosis of neurotransmitters. The effects of LPHN1 activation on exocytosis have been described on several model systems, such as cultured hippocampal neurons and neuromuscular junction (NMJ) using its well known agonist LTXN4C, and include a massive increase in exocytosis characterised by periods of intense release (bursts) interspersed with periods of moderate activity (interburst intervals). However, the molecular mechanisms underlying these effects were yet to be determined. Based on previous observations that LPHN1 is associated to G proteins, and that its activation leads to activation of PLC and increased IP3, we hypothesize that LPHN1 controls exocytosis via the Gαq protein pathway, whose activation ultimately results in the release of Ca2+ from IP3-sensitive Ca2+ stores. Using a pharmacological approach and the current clamp method at the mouse NMJ, we first used LPHN1 KO preparations to study the role of LPHN1 on spontaneous exocytosis in resting conditions, and to show that LPHN1 is the only receptor mediating the effects observed upon stimulation by LTXN4C. Then, we interfered with several molecules involved in the Gαq pathway to test their involvement in LPHN1 activation, and we investigated the role of store-operated (SOCCs) and voltage-gated (VGCCs) Ca2+ channels in mediating the Ca2+ influx that is necessary for the development of LPHN1 effects. Our results support the hypothesis LPHN1 is involved in the regulation of spontaneous exocytosis at rest and that it is the receptor mediating the increased exocytosis following stimulation by LTXN4C; the suggestions that Gαq and its intracellular pathway mediate the effects of LPHN1 activation on spontaneous exocytosis, and that SOCCs and VGCCs (particularly Cav2.1) mediate the Ca2+ influx necessary for the development of LPHN1 effects are also supported by our findings. Altogether, this work uncovered the mechanisms by which G protein-coupled receptors, in this case LPHN1, can regulate the rate of spontaneous neurotransmitter release at the mouse NMJ.

An estimated 2% of the human genome encodes for proteolytic enzymes. It is becoming increasingly apparent that serine proteases have diverse and critical roles in many physiological and pathophysiological processes. Studies investigating the role of serine proteases in the kidney have focussed primarily on their pro-inflammatory effects and their ability to cleave and activate the epithelial sodium channel (ENaC). P2X receptors, which are structurally very similar to ENaC and have been identified throughout the nephron and in the renal vasculature, are thought to contribute to the regulation of tubular transport mechanism and renal haemodynamics, as well as be involved in several renal pathologies. It is shown here that the serine protease, trypsin, has a significant inhibitory effect on recombinant human P2X3 and P2X7 receptor activity. Moreover, it is shown that trypsin may also have an inhibitory effect on purinergic signalling in the mouse cortical collecting duct. In addition, the single channel activity of P2X receptors expressed on the apical membrane of renal collecting duct epithelial cells is described for the first time. Finally, a novel use of the live-tissue slice method is described and the first direct evidence showing trypsin causes significant morphological changes in renal tubules and medullary microvessels in situ is provided. Collectively, data presented here provides evidence to suggest that serine proteases may contribute to several aspects of renal function that have not previously been explored.

Influenza viruses represent an important public health burden since they cause annual epidemics associated with severe illness and mortality in high-risk populations. Additionally, zoonotic influenza virus infections have potential to produce intermittent pandemics, which have led to millions of deaths globally. However, strategies to prevent influenza severity and spread can be implemented. It is known that antibodies against the haemagglutinin play a key role in protection from influenza virus infection, thus both seasonal and pandemic influenza vaccines aim to elicit such antibodies. Generally, they are directed against haemagglutinin globular head epitopes and are able to neutralize closely related influenza strains, but recently antibodies able to neutralize multiple influenza strains and subtypes have also been described. The discovery of these antibodies, primarily directed against the haemagglutinin stalk, has generated interest in understanding how they are generated and how widespread they are in the human population. Furthermore, eliciting such antibodies has become the aim of many ‘universal’ vaccine approaches. However, the study of these cross-reactive antibodies using classical serological assays is problematic since the current assays have been shown to be relatively insensitive in detecting them. The main objective of this thesis was to study the presence and breadth of cross-reactive neutralizing responses in human populations. To overcome the limitations of current serological tests in detecting these responses, lentiviral pseudotype particles bearing the haemagglutinins of different influenza A subtypes and influenza B strains were used as surrogate antigens in neutralization assays. After the generation of these novel tools and the establishment of appropriate controls, pseudotype particle neutralization assays were employed to investigate cross-reactive antibody responses in pre- and post-vaccination sera. Next, the use of chimeric haemagglutinins, in which the globular head was substituted with the head of a different subtype, was incorporated into the pseudotype system. This allowed the differentiation between haemagglutinin head-directed and stalk-directed antibody responses. The ability to efficiently detect broadly neutralizing antibody responses, including those directed against the haemagglutinin stalk, shows that pseudotype particles are tools that should be further characterised and implemented to be used in sero-epidemiological studies and for ‘universal’ vaccine immunogenicity studies.

Xanthine oxidoreductase (XOR) is one of the main purine catabolising enzymes which converts hypoxanthine into xanthine and further into uric acid. The enzyme has a homodimeric structure and contains two FeS centres, one FAD molecule and one molybdenum atom per monomer. Recent evidence clearly demonstrated that XOR activity is highly increased in human hematopoietic cells of myeloid lineage during their pathogen-induced and endogenously generated biological responses. The integrative signalling role and especially involvement of XOR in cross-talk of metabolic and signalling machinery of human leukocytes remains poorly understood. We have demonstrated that XOD is activated in human myeloid cells in response to pro-inflammatory and growth factor stimulation. Hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP1) transcription complexes were found responsible for maintaining XOR catalytic activity and protein levels. Importantly, the mammalian target of rapamycin (mTOR), a major myeloid cell translation regulator, appeared to be essential for XOR activation. Specific inhibition of XOR led to an increase in intracellular AMP levels triggering downregulation of mTOR activation. Taken together, these results show that XOD is not only activated by pro-inflammatory stimuli or SCF (growth factors), but also plays a crucial role in maintaining mTOR-dependent translational control during the biological responses of hematopoietic cells of myeloid lineage. Findings reported in this thesis open a new field in human myeloid cell research and translational medicine. XOR is an easily accessible therapeutic target, which could be pharmacologically corrected using non-toxic drugs.

The main aim of this project was to develop, formulate, characterise and optimise novel pre-formed thin polymer film that will deliver therapeutically relevant drugs via the buccal mucosa route of paediatric patients, using OME as model drug. The development focused on obtaining formulations with optimized drug loading, drug release and permeation, stability and low toxicity. Five different film forming polymers hydroxypropyl methylcellulose (HPMC), methylcellulose (MC), sodium alginate (SA), carrageenan (CA) and metolose (MET) were used initially and subsequently with polyethylene glycol (PEG 400) as plasticiser and L-arg (to stabilise OME). Polymeric gels (1% w/w) were prepared using water and ethanol (10% v/v and 20% v/v) as the casting solvents with PEG 400 at different concentrations (0 and 0.5 % w/w) and the films were obtained by drying the gels in an oven (40 °C). SA and MET films were chosen for drug loading and further investigation (OME stabilisation). These films showed a good balance between flexibility and toughness required for ease of transportation and patient handling. Drug loaded gels showed that OME was unstable, with gels turning red after 20 minutes and therefore required addition of L-arg. From the results obtained, plasticised (0.5 % w/w PEG 400) MET films prepared from ethanolic (20% v/v) gels and containing OME : L-arg ratio of 1:2 showed the most ideal characteristics (transparency, ease of peeling and flexibility) and was the formulation of choice for further investigation. Results obtained for the hydration and in vitro mucoadhesion studies showed that plasticised films had higher swelling capacity and mucoadhesivity than unplasticised films. In addition, BLK films showed higher swelling index and adhesion than DL films, whilst gelatine equilibrated with PBS showed higher values compared with simulated saliva (SS). Dissolution data from optimised DL MET films showed OME release was sustained over 1 hour. Fitting the release data to kinetic models showed that the Korsmeyer-Peppas equations best fit the dissolution data for both PBS and SS media. The permeability profile of optimised DL film using pig buccal tissue, showed that the amount of OME permeating over 2 hours was 275ug/cm2 suggesting that pig buccal membrane is generally quite permeable and also that the OME is released from the films. Application of SCF caused significant changes to the functional and physical properties of the MET films and converted the original DL MET films from a sustained release formulation (1 hour) to a rapid release system, releasing > 90% of OME within 15 minutes and the release of OME from these films followed Higuchi kinetic model. Finally, incorporation of β cyclodextrin (βCD) into DL MET films containing OME:L-arg 1:1, improved the stability of the drug over 28 days under ambient conditions compared to 14 days for the corresponding DL MET films containing only L-arg at a higher loading (OME: L-arg 1:2). The optimised formulations have potential as paediatric buccal delivery system for OME.

The central aim of this thesis is to investigate how physicians, working for Pharmaceutical Product Development (PPD), a clinical research organisation (CRO), make decisions for a new industry standard for good clinical practice in medical device trials. This topic is introduced via review of decision theory and decision-making (DM) in contextual environments. Physician's career experiences, insights and perceptions of how they make regulatory compliance decisions, and how they think these new requirements should be met, are explored in the main study. The research rationale relates to the author's experience of physician DM in non-medical settings during 25 years working in the field, with a desire to ascertain how compliance influences are identified, assessed and synthesized into decisions within the workplace. Furthermore PPD physicians hold senior positions and new industry regulations require regulatory compliance decisions to be made at the highest level. In this study an interpretive phenomenological paradigm was used to ascertain how physicians make sense of industry regulation then make compliance decisions based on their roles, experiences, cues and sources of data available. Literature review identified 4 core themes (decision-making, errors, situation awareness and new requirements) that guided qualitative data collection via 2 mini-focus groups (n=3 per group) and semi-structured interviews (n=12). Review of 18 physicians' data occurred via framework analysis then comparing between contrasting positions presented. The findings found 4-5 dimensions under each core theme from which 2 frameworks were constructed: firstly, using DM tenets to guide physicians' DM in context and, secondly, identifying how to comply with new industry requirements. This research contributes to academia and practice via framework generation for DM in context. It is unique in its contextual exploration, analysis and interpretation of physicians' impressions, from departmental heads to company board members, in relation to their everyday working lives and the decision approaches used to ensure regulatory compliance within their organisational area of responsibility. The thesis ends by considering potential areas for further research such as deploying each framework, applying the framework concepts with other industry legislation changes or exploring alternative research paradigms in PPD.

It is virtually impossible for a practising doctor to sustain the mass of detailed information which is now available on drug interactions. To overcome this problem, a simple, portable drug interaction warning system, the Drug Disc, has been developed. It consists of a reversible unit of two concentric superimposed discs which pivot freely about their common centre. Interactions are indicated by symbols which appear in a window cut into the upper disc when individual drugs or drug categories shown on the two discs are brought into alignment. The information presented embraces the majority of drug interactions which have a bearing on therapeutics and different symbols are used to grade interactions according to their degree of clinical significance. This grading was based on the nature and severity of the interaction, the adequacy of published information, and opinions expressed by the developers of the Disc, including a Working Party established by the Scottish Home and Health Department, and 450 doctors and pharmacists who participated in a trial of the Disc in the United Kingdom. This survey of attitudes about the warning system indicated that the majority of participants found the information provided to be both clinically useful and informative. Subsequent consumer research in other countries has confirmed that the system would be of value both as a practical aid in prescribing and in teaching. The degree of interest and enthusiasm stimulated by the Drug Disc showed that drug interactions are an aspect of modem medicine that cannot be ignored, and suggested that this kind of aid to prescribing was warranted* It is planned to effect a free distribution of the Disc to Health Service Doctors in the United Kingdom and the Excerpta Medica Foundation has accepted responsibility for its worldwide distribution. Further experience with drug combinations and the introduction of new drugs will largely dictate much of the relevance of drug interactions in the future. The Drug Disc will be subjected to constant review and appropriate changes made whenever necessary. It is hoped that a system of this kind will foster a more critical approach to multiple drug therapy and help to reduce many of the hazards implicit in drug combination.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciencias da Saude / Made available in DSpace on 2012-10-16T06:51:37Z (GMT). No. of bitstreams: 0 / O sistema baseado em conhecimentos foi desenvolvimento em dois módulos. O módulo de triagem e o de classificação. O módulo de triagem é construído adaptando o questionário de Fernandes (1993), elaborado e validado em Porto Alegre, através de um inquérito populacional. Este módulo de classificação utiliza as técnicas de inteligência artificial simbólica, com a programação orientada a objetos, usando shell Kappa, classificando as crises epilépticas baseada nos critérios da ILAE "International League Against Epilepsy" (1981). A estrutura do sistema assemelha-se ao desenho dos estudos epidemiológicos populacionais realizados em dois estágios. O sistema foi testado e demonstrou-se um bom desempenho. Os aspectos atuais de avaliação e as implicações ético-legais também foram discutidos. O sistema implementado pode ser útil na pesquisa clínico epidemiológica, no ensino da epilepsia e também pode servir de base para o desenvolvimento de outros sistemas.

Dengue virus (DENV) is one of the most important human pathogens among the genus flavivirus, with 3.9 billion people at risk of infection through mosquitoes, such as the widely spread ‘Asian tiger’ mosquitoes, and the four serotypes of DENV are endemic in over 100 countries in tropical and subtropical regions. Clinical manifestations of infection with DENV range from flu-like symptoms to the life-threatening dengue haemorrhagic fever. The dramatic increase in the incidence of the DENV infection, the rapid spread of DENV to new areas and the recent re-emergence of another member of the genus flavivirus, Zika virus (ZIKV), have highlighted the urgent need for specific antiviral therapies against infections with DENV and related viruses, which are not currently available. DENV RNA-dependent RNA polymerase (RdRp), the enzyme responsible for the synthesis of the viral genome, is one of the most attractive targets for the development of direct acting antiviral agents but its molecular mechanisms are poorly understood. Thefore, the aims of this PhD project were i) to build a model of the de novo initiation complex of DENV RdRp, of which there is currently no crystal structure available, ii) in silico design and synthesis of novel nucleoside and nucleotide analogues as potential inhibitors of DENV replication, iii) and finally to investigate the mechanism of the RNA synthesis by DENV RdRp. Molecular modelling techniques allowed for the creation of a model of the de novo initiation complex. The application of in silico drug design approaches resulted in the identification of three families of promising adenosine analogues: ribose-modified, nucleobase-modified and acyclic adenosine analogues. Strategies for the preparation of these nucleosides were investigated and ten adenosine analogues and eight nucleotide prodrugs, which are phosphoramidate ProTides, of specific nucleosides were synthesised and sent for biological evaluation in vitro. Innovative microwave irradiation conditions for the preparation of phosphoramidate ProTides were developed and successfully applied to synthesised nucleoside analogues. Finally, the application of molecular dynamics simulation methods on different complexes of DENV RdRp provided insights on the conformational changes of DENV RdRp during the synthesis of the viral genome. These results contributed to the understanding of DENV RdRp activity and will aid the design of inhibitors of the viral replication.