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[eng] INTRODUCTION: Antimicrobial resistance is one of the world’s major challenges in both microbiology and public health since infections caused by multidrug-resistant bacteria are reaching alarming levels. The world is currently facing a global antibiotics crisis and some new strategies need to be explored to tackle these resistant infections. The outer membrane is a differential structure of Gram-negative bacteria that works as a highly effective selective permeability barrier. Thus, the permeation through the Gram- negative cell envelope is a challenge for drug compounds to reach their targets. One path to open new antimicrobial perspectives is the chemical modification of old antimicrobial compounds that may result in optimized drugs with improved antimicrobial properties. In this context, the activity exploration of the derivatives of Microcin J25 and Trimethoprim was carried out. HYPOTHESIS: The main hypotheses of this thesis are that chemical modifications of current antibiotics may significantly contribute to overcoming the problems arising from the increase and spread of antibiotic resistance in bacteria. Moreover, the combination of these modified compounds with old-rescued antibiotics may contribute to the solution bases of the problem caused by resistance. Objectives: The main objective of this thesis is to determine the antimicrobial properties of chemically modified drug compounds. The secondary objectives of this thesis are divided in two parts: Microcin J25: Study of the antimicrobial activity of the modified Microcin J25 as well as determination of its toxicity. Trimethoprim: Study of the antimicrobial activity and cytotoxicity of the Trimethoprim derivatives, exploration of the effect of the new derivatives on the Dihydrofolate Reductase (DHFR) enzyme and start a computational approach to decipher the intimal mechanisms of action. METHODOLOGY: The antimicrobial activity of the derived compounds was explored against planktonic bacteria (studying the Minimum Inhibitory Concentration and the FIC index) and against sessile bacteria (exploring the Minimum Biofilm Eradication Concentration and the Biofilm Prevention Concentration). The growth curves of several microorganisms in contact with these compounds and in combination with colistin were also studied. The cytotoxicity of all the compounds was tested. An enzymatic assay with E. coli DHFR as well as a docking modelling were carried out to investigate the mechanism of action of TMP derivatives. RESULTS: With respect to Microcin MccJ25, it has been detected that the chemical modification of the compound resulted in a new peptide without antimicrobial activity. It acted synergistically with sublethal concentrations of colistin. When referring to Trimethoprim (TMP), some of the new derivatives showed antibacterial similar to that of TMP. Moreover, almost all the new TMP-like compounds acted synergistically with SMX. P. aeruginosa PAO1 was fully resistant to TMP and all its derivatives as well as to the combination of TMP-SMX. The combination of TMP, TMP- like molecules and SMX with colistin enhanced their antimicrobial efficacy against E. coli, P. aeruginosa and S. marcescens. Compounds 1a and 1b, like TMP, strongly inhibited the activity of the E. coli DHFR. Additionally, it was detected that the heterocyclic ring of the compound 1a fills the pocket occupied by the nicotinamide ring of NADPH. CONCLUSIONS: The present PhD thesis has led to some relevant conclusions. With respect to Microcin MccJ25, the chemical modification of the compound avoided its detection by the membrane receptor FhuA. Moreover, it did not affect the interaction with the target and, as the polymyxin facilitated the microcin entrance across the membrane, once inside the cell, the new compound retained its ability to inhibit the growth of bacteria. When referring to Trimethoprim (TMP), the derivatives showed interesting antimicrobial activities acting synergistically with SMX. The combination of TMP, TMP- like molecules and SMX with colistin enhances their antimicrobial efficacy by permeabilizing the cells. Compounds 1a and 1b, like TMP, strongly inhibited the activity of the E. coli DHFR and it was suggested that both molecules interact with the analogues during inhibition. The search of new antimicrobial compounds is one of the main pathways to overtake bacterial resistance to antibiotics. All putative compounds should be tested in conditions in which outer membrane role as permeability barrier is inactivated. Their assay together with sublethal concentrations of colistin is proposed as one of the methods of election., [spa] INTRODUCCIÓN: La resistencia a los antimicrobianos es uno de los principales desafíos del mundo tanto en microbiología como en salud pública, ya que las infecciones causadas por bacterias multirresistentes están alcanzando niveles alarmantes. Una opción para abrir nuevas perspectivas antimicrobianas es la modificación química de compuestos antimicrobianos ya existentes que pueden resultar medicamentos optimizados con propiedades antimicrobianas mejoradas. En este contexto, en esta tesis se llevó a cabo la exploración de la actividad de los derivados de Microcina J25 y Trimethoprim. HIPÓTESIS: Las principales hipótesis de esta tesis son que las modificaciones químicas de los antibióticos actuales y la combinación de estos compuestos modificados con antibióticos antiguos rescatados pueden contribuir significativamente a superar los problemas que surgen del aumento y la propagación de la resistencia a los antibióticos en las bacterias. Objetivos: El principal objetivo de esta tesis es determinar las propiedades antimicrobianas de compuestos químicamente modificados. Metodología: Se exploró la actividad antimicrobiana de los compuestos derivados frente bacterias planctónicas y frente bacterias sésiles. También se estudiaron las curvas de crecimiento de varios microorganismos en contacto con estos compuestos y en combinación con colistina. Se ensayó la citotoxicidad de todos los compuestos. Se llevó a cabo un ensayo enzimático con E. coli DHFR así como un modelo de docking. RESULTADOS Y CONCLUSIONES: Con respecto a la Microcina MccJ25, se detectó que la modificación química del compuesto dio como resultado un nuevo péptido sin actividad antimicrobiana ya que se evitó su detección por el receptor de membrana FhuA. Esta actuó de forma sinérgica con concentraciones subletales de colistina debido a que la polimixina facilitó la entrada de la microcina a través de la membrana y, una vez dentro de la célula, el nuevo compuesto conservó su capacidad para inhibir el crecimiento de las bacterias. En cuanto a Trimethoprim (TMP), algunos de los nuevos derivados mostraron un efecto antibacteriano similar al de TMP. Además, casi todos los nuevos compuestos actuaron sinérgicamente con SMX. P. aeruginosa PAO1 fue totalmente resistente a TMP y todos sus derivados, así como a la combinación de TMP-SMX. La combinación de TMP, análogos de TMP y SMX con colistina mejoró su eficacia antimicrobiana contra E. coli, P. aeruginosa y S. marcescens ya que ésta permeabilizó las células. Los compuestos 1a y 1b, como TMP, inhibieron la actividad de la DHFR de E. coli. Además, se observó que el anillo heterocíclico del compuesto 1a llena el bolsillo ocupado por el anillo de nicotinamida de NADPH y se sugirió que ambas moléculas interactúan con los análogos durante la inhibición.

Dissertação de mestrado em Gestão de Unidades de Saúde, Este estudo tem como objetivo avaliar os níveis de ruído no ambiente do serviço de urgência e analisar o impacto deste na qualidade de vida e motivação dos profissionais de enfermagem (incluindo SUB Monção e SUB Ponte de Lima). Este estudo carateriza-se como quantitativo, exploratório e descritivo e baseou-se na aplicação de um questionário online como instrumento de recolha de dados e na utilização de um sonómetro para medir os níveis de ruído nas variadas áreas do SU. Dos resultados obtidos é de realçar que os valores de ruído no SU situam-se acima dos valores recomendados quer para o conforto acústico quer para a saúde ocupacional, constituindo-se por isso um risco para os profissionais de saúde. Os profissionais inquiridos neste estudo identificaram o serviço de urgência como ruidoso, principalmente no turno da manhã, referindo sintomas como irritabilidade, stress, cefaleias, cansaço fácil, dificuldade na comunicação e concentração. Mediante o desafio relacionado com o controlo de ruído em cenário de urgência/emergência, a presente pesquisa concluiu que apesar de ser do conhecimento generalizado dos profissionais a existência de ruído, das fontes e dos efeitos nefastos do mesmo sobre a qualidade de vida dos profissionais, trabalho e motivação ainda não se conseguiram implementar medidas de forma a manter os níveis de pressão sonora dentro dos limites estimados pelas organizações internacionais. Os resultados indicaram a necessidade de desenvolver programas de sensibilização e educação para a saúde que visam fundamentalmente a consciencialização de todos os profissionais, não só do SU, para a necessidade de modificarem e ajustarem comportamentos à nova prática do cuidar em enfermagem., This study aims to assess the noise levels in the emergency room environment and analyze its impact on the quality of life and motivation of nursing professionals (including SUB Monção and SUB Ponte de Lima). This study is characterized as quantitative, exploratory and descriptive and was based on the application of an online questionnaire as a data collection instrument and on the use of a sound level meter to measure noise levels in the various areas of the SU. From the results obtained, it is noteworthy that the noise values in the SU are above the recommended values for both acoustic comfort and occupational health, thus constituting a risk for health professionals. Professionals interviewed in this study identified the emergency room as noisy, especially in the morning, reporting symptoms such as irritability, stress, headaches, easy tiredness, difficulty in communication and concentration. Given the challenge related to noise control in an urgent/emergency setting, this research concluded that despite the widespread knowledge of professionals about the existence of noise, its sources and harmful effects on the quality of life of professionals, work and motivation measures to maintain sound pressure levels within the limits estimated by international organizations have not yet been implemented. The results indicated the need to develop health awareness and education programs aimed fundamentally at raising the awareness of all professionals, not just the ER, of the need to modify and adjust behaviors to the new practice of nursing care.

In this project, the interaction between the fitopathogen fungus Ustilago syntherismae and the plant at which it infects, Digitaria sanguinalis, has been studied. U. syntherismae has a drastic effect on the reproduction of D. sanguinalis because it causes an important reduction in the spikelet production. This fact is because the smut is formed in the plant's inflorescence replacing spikelets by a spore mass. This project has been mainly focused on a stage which is considered essential in the knowledge of the pathosystem: the infection phase. An experiment was made in order to determine if the virulence of the fungus during infection phase varies according to the spikelet and spore type which are interacting. In addition, it was studied whether the germination pattern (radicular or coleoptilar) had an effect in the infection grade. Two types of spikelets were used; some came from apparently disease-free plants (carrying only spikelets) and the others came from partly smutted plants (carriers of splikelets and spores). Furthermore, two types of fungus spores were also used; some came from completely smutted plants (carriers of only spores) and some from partly smutted plants. A vacuum infiltration method was used to facilitate the in vitro contact of spikelets and spores in order to induce the interaction between the plant and the fungus artificially. Inoculated seedlings were dyed and observed with the optical microscope to verify the existence of fungus in their tissues. The experiment was divided into two assays: one with material collected in 2009 and the other with material collected in 2011. These two assays showed that significant differences (P

New advances have been released in textile industry. Contributions at the level of textile fiber chains engineering has allowed modification of their structure, production of smart polymers responding to changes in environment, and attachment or entrapment of cells and bioactive molecules. Likewise, our society high demand of hygienic patterns, has raised the intensive research and development of antimicrobial textiles. Applications are being extended to underwear, sportswear, home furnishing, protective clothes, wound-dressings and in areas with high risk of microbial infection, as hospitals, schools and hotels. Throughout last decades hospitals have faced tough challenges concerning microbial multi-resistance, especially in immunodepressed patients. The strongest cause for microbial resistance may be due to the abuse of antibiotics uptake, either by humans to treat something non appropriately, as by animals, to earn weight. The development of antimicrobial textiles arise as a promising solution that may significantly decrease the risk of nosocomial infections. Several antimicrobial agents have been applied in textiles, namely quaternary ammonium compounds, silver, polyhexamethylene biguanides and triclosan. However, they have shown a reduced spectrum of microbial inhibition which cause resistances, cytotoxicity causing skin irritation, as well as toxic to the environment. Furthermore, these compounds gradually lose their bioactivity with use and launderings. L-cysteine (L-Cys) that is found in several living organisms is a natural defensive thiolated aminoacid never studied before as a potential antimicrobial agent for textiles, which can grant antibacterial properties without cytotoxicity. Furthermore, antimicrobial peptides (AMPs) belong to innate immune system of multicellular organisms and appear as an alternative to antibiotics. They are small, amphipathic, and strongly cationic which bind to negatively charged phospholipid headgroups of microbial membranes. Although the mechanism of AMPs-microbial killing is still not known, many hypotheses have been proposed: (i) membrane depolarization, (ii) formation of physical holes at the membrane, (iii) programmed bacterial death processes, (iv) phospholipidic redistribution, and (v) internalization of the AMP. They have broad-spectrum antimicrobial activity. Once their target is the bacterial membrane microorganisms hardly develop resistance, otherwise they would have to change all their lipidic composition and/or organization, which is high demanding and not energetically worth it. During this work, non-covalently adsorbed L-Cys to wool (patent PAT 104540 A) and to cotton showed to be non-toxic to human cells, and had antimicrobial effects against Gram-negative and Gram-positive bacteria and its main mechanism of action on cotton was assessed by flow citometry. Antimicrobial peptides (AMPs) will also be immobilized on textiles, in order to find if textile imobilized-AMP can attract and kill bacteria. Natural polymers have shown few adverse reactions, once they have excellent humidity control, biocompatibility and low-allergic responses, due to their similarity to macromolecules which biological environment is prepared to recognize and to deal with metabolically. AMPs will be selected, based on their 3D structure, terminal charge and size. Best-studied AMPs are cationic due to their action on negative surface charged microorganisms. Evaluation of minimal inhibitory concentration (MIC) of AMPs will elucidate the amount of AMPs to be used to functionalize textile substrates and cytotoxicity studies will provide the toxicity of functionalized textiles to human cells. In order to develop long-lasting and washable functionalized textiles we propose the covalent binding of AMPs on textiles through selected chemistries already employed on surface modifying of medical devices elsewhere. Alternatively, we will use plasma treatment, which is usually used to modify many surface properties of polymeric materials. This study may allow the development of innovative antimicrobial textiles, simulating microbial-free microenvironments in order to develop, in the future, antimicrobial fabrics to avoid airborne spreading and improve patient’s quality of life in a hospital context.

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Submitted by Ana Costa (apcosta@porto.ucp.pt) on 2010-09-21T16:31:34Z No. of bitstreams: 1 Flow injection determination of fluoride.pdf: 1146199 bytes, checksum: 69277839286c54d66a8498d6fdf110b8 (MD5) Made available in DSpace on 2011-09-13T16:16:55Z (GMT). No. of bitstreams: 1 Flow injection determination of fluoride.pdf: 1146199 bytes, checksum: 69277839286c54d66a8498d6fdf110b8 (MD5) Previous issue date: 2009

Submitted by Ana Costa (apcosta@porto.ucp.pt) on 2010-09-21T14:48:35Z No. of bitstreams: 1 Desenvolvimento de uma metodologia de análise.pdf: 680449 bytes, checksum: 109b9bab1a96e8b3743cffa085a68a95 (MD5) Made available in DSpace on 2011-09-13T14:37:48Z (GMT). No. of bitstreams: 1 Desenvolvimento de uma metodologia de análise.pdf: 680449 bytes, checksum: 109b9bab1a96e8b3743cffa085a68a95 (MD5) Previous issue date: 2009