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.

This doctoral thesis reports on a longitudinal, mixed methods investigation of staff and students’ views, expectations, and experiences of a collaborative pharmacy programme between Cardiff University School of Pharmacy and Pharmaceutical Sciences (CU) and Taylor’s University School of Pharmacy (TU). Despite a growing body of empirical research on transnational staff and students’ expectations and experiences, longitudinal mixed methods studies are rare. This study combined a qualitative interview-based and focus group approach with a quantitative questionnaire-based method. The overall aim is to gain a better understanding of the teaching and learning experiences of staff and students in a transnational education (TNE) programme. The qualitative element explored staff expectations and experiences in the early stage of the collaborative programme while student expectations and experiences were investigated at different points in time throughout their 4-year pharmacy study. The quantitative element investigated and compared the learning environment perceived by participating students in TU and CU. Data collection took place over a period of 36 months and comprised four phases. In Phase 1, staff and students’ initial expectations and experiences of a new collaborative pharmacy programme were explored using staff interviews and student focus groups. In Phase 2, a sample of students from CU and TU were recruited to participate in a questionnaire study to assess students’ perceived learning environment. In Phase 3, a number of studies were carried out using focus groups in order to find out students’ pre-arrival expectations and post-arrival experiences. Phase 4 involved a self-administered questionnaire with graduate students to assess students’ opinions about their overall experiences at the universities. The study revealed staff and students' expectations and their actual experiences in relation to the delivery of a transnational education. It was found that those students who participated were able to cope with sociocultural adjustment in a new learning environment. The study also provided indications of the need for training and professional development for staff to teach in a transnational environment. Finally, Malaysian students who come from a teachercentred pedagogy background should be informed and trained earlier before their transfer to lessen the impact brought about by intercultural differences in teaching and learning.

Central obesity is a complex cardiometabolic entity strongly linked to the constellation of risk factors such as insulin resistance, hypertension, dyslipidaemia and physical inactivity, which when combined lead to an increased risk of type 2 diabetes and cardiovascular disease. The available evidence suggests that these conditions are linked to microvascular dysfunction, which may appear much before the onset of overt cardiovascular and metabolic disease. However, in apparently healthy but viscerally obese subjects, little is known about the interactions between cardiometabolic risk factors, including microvasculature, which could be potential targets for early therapeutic intervention. Statins are attributed to have pleiotropic properties, but their effects on insulin resistance and microcirculation are still uncertain. The hypotheses for this study were that in centrally obese but non-diabetic subjects: • Skeletal muscle exchange capacity influences levels of HbA1c. • Diminished insulin sensitivity in skeletal muscle is associated with reduced microvascular exchange capacity. • Microvascular functional dilator capacity is independently associated with insulin sensitivity and age. • Six months of treatment with high dose statin improves insulin sensitivity and reverses microvascular dysfunction. • Cardiorespiratory fitness is independently associated with cardiac diastolic function and arterial stiffness. A double-blinded, randomised, placebo controlled trial was conducted in white Caucasians aged 29-69 with abdominal obesity and a cardio-metabolic phenotype. Insulin resistance was assessed by stepped hyperinsulinaemic euglycaemic clamp and fasting insulin sensitivity indices. Microvascular function was examined with venous congestion plethysmography and Laser Doppler Fluximetry. It was demonstrated that in centrally obese, non-diabetic subjects with modest insulin resistance, skeletal muscle exchange capacity was associated negatively and independently with HbA1c, positively and independently of visceral fatness with insulin sensitivity, and that functional dilator capacity was strongly and positively associated with insulin sensitivity and age, independently of each other. Six months of intensive treatment with Atorvastatin did not improve insulin sensitivity or microvascular function. A strong association was shown between cardiorespiratory fitness and measures of diastolic function and arterial stiffness. In conclusion, this thesis presented novel aspects of cardio-metabolic factors and microvascular relationships, which indicate the early onset of microvascular dysfunction in obesity and the importance of fitness in maintaining arterial flexibility and cardiac diastolic function. Atorvastatin has no role in improving insulin sensitivity and reversing microvascular dysfunction.

Staphylococcus epidermidis biofilm formation is a primary cause of medical device infections, which are persistent and difficult to eradicate because biofilms intrinsically exhibit a naturally high level of antibiotic resistance. Although biofilm antibiotic resistance or tolerance is a multifactorial process, some mechanisms such as limited diffusion, low metabolic activity and persister cells, contribute to the failure of antibiotics in the treatment of biofilm infections. Current, antibiotic treatment strategies may provide biofilm infections with intermittent exposure to sub-minimum inhibitory concentrations (sub-MIC) of antibiotics. Biofilms have been shown to display an increase in antibiotic tolerance when exposed to antibiotics at sub-MIC. Such mechanisms of adaptive antibiotic resistance are not well characterized but are of extreme clinical importance. This project showed that exposure to sub-MIC vancomycin increases the virulence of S. epidermidis biofilms because it induces vancomycin tolerance. BODIPY FL-vancomycin (fluorescent vancomycin conjugate) and confocal microscopy were used to show that the penetration of vancomycin through sub-MIC vancomycin pre-treated S. epidermidis biofilms was impeded, when compared to control, untreated biofilms. In addition, the results showed that a wide range of sub-MIC vancomycin concentrations induced an increased amount of extracellular DNA (eDNA) within the matrix of sub-MIC vancomycin treated biofilms. Finally, a set of ex vivo experiments using extracted exogenous S. epidermidis DNA revealed that exogenous S. epidermidis DNA binds vancomycin. Collectively these findings suggest that sub-MIC vancomycin exposure increase the abundance of eDNA in the matrix of S. epidermidis biofilms, which protects the biofilm community from subsequent vancomycin exposure by binding vancomycin as it travels through the matrix. Therefore the work in this project provides details of an eDNA-based mechanism of adaptive antibiotic tolerance in sub-MIC vancomycin treated S. epidermidis biofilms, which might be an important factor in the persistence of biofilms infections.

Hospital discharge is a complex process that can result in errors and delays for patients, particularly around the supply of medicines and communication of information. This programme of work (PoW) aimed to develop an innovative model of care for the supply of medication at hospital discharge to provide safe, quality and effective transfer for patients from hospital to community care. The PoW consisted of four phases which used both quantitative and qualitative approaches. Phase 1 involved semi-structured telephone interviews with 13 Chief Pharmacists. Analysis identified the current discharge process across the range of hospitals as well as key issues and examples of good practice at discharge. Discharge processes were similar across hospitals with issues common to all. Phase 2 used questionnaires to establish patient perceptions of the current discharge process in a large city-centre teaching hospital. The 104 inpatients recruited were 60% (n=62) male, average age was 55 (range 19-93), from both medical and surgical wards. Most patients, 89% (n=87) were satisfied with their hospital discharge but believed it took too long. The perceived main cause of delay was waiting for medicines. Other highlighted issues included limited counselling by pharmacists and a need for more patient involvement at discharge. Phase 3 utilised findings from phases 1 and 2 to inform the development of a new model of care for patient discharge. Phase 4 consisted of semi-structured interviews and focus groups with stakeholders in patient discharge (n=37), to evaluate the proposed new model of care. Stakeholders successfully evaluated the new model, highlighting areas of the new model of care that would work well and where problems may arise. The model of care was refined based on these findings, with the suggestions for overcoming logistical issues considered. The PoW successfully developed an innovative model of care for patient discharge.

Topical drugs are not developed by the same process as oral drugs. The process is more uncertain and contains gaps. This leads to a poor discharge of risks before going to the clinical phases. The topical drug development process is reviewed in the introduction of the thesis. In particular, past and current topical drug development practices are described and compared to the oral drug development process. The large risks taken during the topical drug development are pointed out. These risks are largely associated with a lack of pharmacokinetic's involvement prior to the drug candidate selection stage. Pharmacokinetics is considered after drug selection when it is often too late. Furthermore, the topical pharmacokinetic techniques available appear to be not suitable for three reasons: accessibility to the pharmacokinetic techniques, meaning of the data generated and reliability of these data. It concludes that the knowledge of target skin tissue concentration would be key for a more rational drug development process. To this end, the primary objective of this thesis is to define a way of measuring drug concentration in skin tissue after topical application that is reliable, effective and practical. A secondary objective is then from the knowledge of the skin tissue concentration, to develop a topical PharmacoKinetic/PharmacoDynamic model to predict likely efficacy for a topical drug candidate. First a direct skin tissue concentration approach is described that brings theoretical reliability into the pharmacokinetic data generated and improves throughput. However the pharmacokinetic data generated have limited use as total drug (bound + unbound) tissue concentration is measured while, pharmacodynamically, only the unbound fraction is of interest. An indirect skin tissue concentration determination is then proposed. It consists in predicting the in vivo unbound drug concentration in diseased skin tissues. Three steps are required: In the first step, the in vitro percutaneous flux is linked with the unbound drug concentration in the dermis. From there, the in vivo unbound drug concentration in all the skin tissues is defined using different physiological parameters. Finally, taking into account the effect of the skin disease on skin permeability and dermal capillary clearance, the in vivo unbound drug concentration in skin tissues in diseased skin is defined. The predicted concentration is therefore calculated from a constant (which is skin disease dependent) and from the in vitro percutaneous flux (which is an accessible and reliable experimental pharmacokinetic data). A PharmacoKinetic/PharmacoDynamic model is then built. This model delivers two types of information: -1- The "efficacy index" which is a prediction of efficacy for a drug candidate based on percutaneous flux and drug potency and -2- the "systemic safety index" which is an assessment of systemic exposure based on total systemic clearance and plasma protein binding. To check the validity of this new model, a validation exercise is run with the key eight topical drugs classes: NSAIDS, anaesthetics, retinoids, corticosteroids, vitamin D3 derivatives, antifungals, antibacterials for acne and immunomodulators. For seven out of the eight classes, the validation of the model is good. For the last class, the antibacterials for acne, the model underpredicts efficacy and it is suggested that the route of entry of antibacterial agents in acne occurs via the sebaceous duct as opposed to the more classic stratum corneum pathway. Finally, three pilot studies are conducted with the aim to improve the quality and relevance of the data generated with in vitro percutaneous flux studies as well as the access to this technique and throughput of this technique.

The existence of polymorphic forms of drug substances has implications for therapeutic performance, handling and storage. This study investigates the development of a novel approach to surface analysis of drug polymorphs, with the aim of extending the capabilities of this approach to perform real time analysis of polymorphic transformation during pharmaceutical product development. This was achieved here, using diffuse reflectance visible spectroscopy (DRVS) and the colour change which occurs when pH indicator dyes are deposited on the surface. The pH indicators used were phenol red (PR), thymol blue (TB) and methyl red (MR). Two polymorphs each of indomethacin (IMC), carbamazepine (CBZ), caffeine (CFN), sulfanilamide (SFN) and furosemide (FRS) were examined. The interaction of the adsorbed dye with each of the polymorphs showed different behaviour, manifested by different colours. An analysis of the crystal structures and the acid/base properties of the drug molecules provided a rationalisation for the different colours exhibited by the polymorphs‘ surfaces. The least stable form of each polymorphic pair studied showed more extensive interaction with the adsorbed dye molecules. Observed colour reveal underlying differences at a molecular level between the surfaces of pairs of polymorphs. The different colours exhibited by the indomethacin polymorphs were further examined using hygroscopicity studies, contact angle measurements and computer simulation. The contact angles of several liquids with the polymorph surface were measured in order to characterise the nature of the functional groups exposed on the surface of the polymorphs. The surface structure and external morphologies of polymorphs were predicted by molecular modelling using the attachment energy model. The predicted morphology was confirmed by scanning electron micrographs (SEM) and the miller index of the dominant face was confirmed by X-ray powder diffraction (XRD). Results revealed that although the surfaces of both polymorphs are largely hydrophobic, the metastable form- IMC-α has a greater number of polar functional groups on the surface. Further measurements were carried out using DRVS and adsorbed TB to study the kinetics of the solid-state transformation of SFN- to SFN-. The rate of transformation was followed at 128ºC by monitoring the ratio of the two DRVS bands at 454 nm and 604 nm. The kinetic data was analysed using sixteen solid-state kinetic models to obtain the best fit. The thermally induced polymorphic transformation of the SFN-β (particle size of ≥ 450μm) can be best described by the first order kinetic model (R2 = 0.992) with a rate constant, k of 2.43 x 102 s-1. The DRVS instrument used herein is not adapted for in situ studies; however, because of its non-destructive interaction with the sample and rapid data collection time of 5s per spectrum, it does offer considerable potential as a tool for real time monitoring of polymorphic transformation.

A preocupação com a qualidade ambiental aumentou significativamente nos últimos anos. Isso é evidente pela rígida legislação ambiental e pela mudança de comportamento da sociedade frente a este assunto. Nesse contexto os estabelecimentos de serviço de saúde estão sendo obrigados a se adequarem aos novos sistemas de gerenciamento de resíduos sólidos, oriundos das suas atividades, mas nada se comenta à respeito do efluente gerados por estes empreendimentos. Os problemas associados aos efluentes gerados nos centros de serviços de saúde tem sido motivo de preocupação devido ao desconhecimento do perigo potencialmente escondido neste. Na cidade de Porto Alegre as águas residuais provenientes dos hospitais não são tratadas, sendo transportadas por redes coletoras até o Lago Guaíba. Este é uma das principais fontes de abastecimento de parte da população desta cidade. Este trabalho apresenta como objetivo analisar as características básicas físico-químicas de efluentes hospitalares de quatro unidades de saúde da cidade de Porto Alegre – RS, comparando-os com a legislação vigente no que diz respeito a limites de descarte de efluentes. A amostragem proposta foi aplicada a quatro unidades paralelamente, sendo coletadas cinco amostras de cada unidade num período de dois anos. As amostragens foram compostas. Por fim, constatou-se que os efluentes oriundos de serviço de saúde, para a segurança, principalmente do Meio Ambiente, deverão ser previamente tratados antes de atingir a rede pública coletora.

Este trabalho avaliou o lançamento de efluentes líquidos de quatro hospitais, de diferentes especialidades médicas, no município de Porto Alegre – RS, Brasil. Foi realizada uma pesquisa previa, nas especialidades de Traumatologia, Pneumologia, Oncologia e Geral, a fim de obter dados referentes ao descarte dos respectivos efluentes. Após foram realizadas cinco amostragens de cada especialidade hospitalar durante um ano. Os parâmetros determinados seguiram a Portaria Nº 05/89 – SSMA que aprova a Norma Técnica SSMA Nº 01/89 – DMA, que dispõe sobre critérios e padrões de efluentes líquidos a serem observados por todas as fontes poluidoras que lançam seus efluentes nos corpos d’água interiores do Estado do Rio Grande do Sul. Dos trinta e cinco parâmetros avaliados em cada amostragem, para cada hospital, vinte e oito apresentaram, concentração dentro do limite de descarte determinado pela Portaria 05/89, e sete ultrapassaram os limites de concentração da mesma Legislação. O consumo médio de água e a geração de esgotos, relacionados ao número de leitos, aproximase com os valores apresentados nas referências bibliográficas, considerando que, o número de atendimentos influencia diretamente na geração de efluentes hospitalares. A relação entre os resultados de DBO5 e DQO, de todas as especialidades, indica semelhança aos efluentes domésticos. Os resultados das concentrações de DBO5, DQO, Nitrogênio Total, Fósforo Total, pH, Coliformes Fecais e Coliformes Totais, tratados estatisticamente, apresentam semelhanças entre as amostragens e as especialidades comprovando que não há diferenças significativas entre efluentes líquidos nas especialidades hospitalares estudadas.

A cidade de Porto Alegre, devido à localização, abriga espécies vegetais procedente de dois corredores principais: o corredor Atlântico e o corredor do Alto Uruguai. Os morros graníticos sobressaem-se na paisagem porto-alegrense abrigando matas nas suas encostas sul e campos nos topos e encostas nortes, devido às condições criadas pela exposição solar diferenciada. O Morro Santana, com 311 metros é o mais alto deles. Com o objetivo de caracterizar a composição e a estrutura e aspectos da dinâmica de regeneração desta mata, é realizada amostragem do componente arbóreo-arbustivo, dividindo-se os indivíduos em três componentes, de acordo com suas medidas de alturas e de DAP (diâmetro à altura do peito): Componente 3 (0,20 m ≤ h < 1 m); Componente 2 (h ≥ 1 m e DAP < 3 cm) e Componente 1 (DAP≥ 3 cm), sendo que, para a estimativa de regeneração natural por classes e total (RN e RNT), a qual é dada em porcentagem, os componentes 2 e 3 foram distribuídos em três classes de altura: Classe 1 (0,20m ≤ h < 1m); Classe 2 (1m ≤ h < 3m); Classe 3 ( h ≥ 3m e DAP < 5cm).O método utilizado é o de parcelas de 100 m2 (Componente1), 25 m2 (Componente 2), e 4 m2 (Componente 3). Através do software MULVA 5, foram realizadas análises de agrupamento e ordenação (PCoA) para os três componentes. O levantamento resultou em: 505 indivíduos, pertencentes a 63 espécies, 51 gêneros e 30 famílias no Componente 1; 470 indivíduos, distribuídos em 44 espécies, 33 gêneros e 19 famílias no Componente 2; 191 indivíduos, distribuídos em 30 espécies, 26 gêneros e 18 famílias no Componente 3. No Componente 1 destacam-se Guapira opposita (Nyctaginaceae), Pachystroma longifolium (Euphorbiaceae) e Eugenia rostrifolia (Myrtaceae). Nos Componentes 2 e 3 destacam-se Psychotria leiocarpa e Faramea montevidensis (Rubiaceae), Mollinedia elegans (Monimiaceae) e Gymnanthes concolor (Euphorbiaceae). A composição florística nos três componentes, em ordem decrescente de importância, foi dada por espécies de ampla distribuição, espécies Atlânticas e espécies do Alto Uruguai. As espécies secundárias perfizeram a maioria absoluta da amostra nos três componentes, sendo que as secundárias tardias superaram as secundárias inicias. As espécies pioneiras foram infimamente representadas nos três componentes inventariados. Quanto às síndromes de dispersão, mais de 80 por cento das espécies de ambos os componentes apresentou síndrome zoocórica. O restante das espécies apresenta as síndromes autocórica e anemocórica em proporções semelhantes.A análise fitossociológica indicou tratar-se de uma mata baixa, sem estratificação definida, com dominância de um número reduzido de espécies de ocorrência comum na região, sendo que a grande maioria das espécies conbribui com um ou dois indivíduos. A diversidade específica (H’) foi estimada em 3,30 (Componente 1); 2,81 (Componente 2) e 2,86 (Componente3) e a equabilidade (J’) em 0,80 (Componente 1) ; 0,74 (Componente 2) e 0,84 (Componente 3). As análises de agrupamento e de ordenação não evidenciaram diferenças entre as unidades de amostragem da borda e aquelas do interior da mata. Os maiores valores na estimativa de regeneração natural total (RNT) foram concentrados pelas espécies de sub-bosque com maiores densidades e freqüência (Psychotria leiocarpa, Faramea montevidensis, Mollinedia elegans e Gymnanthes concolor). Os representantes que compõem o dossel da mata com maiores valores de regeneration foram: Guapira opposita, Eugenia rostrifolia e Cupania vernalis (Sapindaceae). Os resultados da estimativa de regeneração natural indicaram que a maioria das espécies parece estar obtendo sucesso quanto ao recrutamento de novos indivíduos, o que remete a um bom estado de conservação da mata, a qual, permanecendo as condições atuais, deverá no futuro manter uma composição florística semelhante a atual.