Study of membrane emulsification process as a pre-step for the microencapsulation of lipid compounds by spray drying

Dissertation presented to Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa for obtaining the master degree in Membrane Engineering

Bilirubin directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat mitochondria

Background/Aims: Unconjugated bilirubin (UCB) impairs crucial aspects of cell function and induces apoptosis in primary cultured neurones. While mechanisms of cytotoxicity begin to unfold, mitochondria appear as potential primary targets. Methods: We used electron paramagnetic resonance spectroscopy analysis of isolated rat mitochondria to test the hypothesis that UCB physically interacts with mitochondria to induce structural membrane perturbation, leading to increased permeability, and subsequent release of apoptotic factors. Results: Our data demonstrate profound changes on mitochondrial membrane properties during incubation with UCB, including modified membrane lipid polarity and fluidity (P , 0:01), as well as disrupted protein mobility(P , 0:001). Consistent with increased permeability, cytochrome c was released from the intermembrane space(P , 0:01), perhaps uncoupling the respiratory chain and further increasing oxidative stress (P , 0:01). Both ursodeoxycholate, a mitochondrial-membrane stabilising agent, and cyclosporine A, an inhibitor of the permeability transition, almost completely abrogated UCB-induced perturbation. Conclusions: UCB directly interacts with mitochondria influencing membrane lipid and protein properties, redox status, and cytochrome c content. Thus, apoptosis induced by UCB may be mediated, at least in part, by physical perturbation of the mitochondrial membrane. These novel findings should ultimately prove useful to our evolving understanding of UCB cytotoxicity.

How to Measure the Deterrence Effects of Merger Policy: Frequency or Composition?

We show that the number of merger proposals (frequency-based deterrence) is a more appropriate indicator of underlying changes in merger policy than the relative anti-competitiveness of merger proposals (composition-based deterrence). This has strong implications for the empirical analysis of the deterrence effects of merger policy enforcement, and potential implications regarding how to reduce anti-competitive merger proposals.

Multy-dimensional composition by objectives in aspect-oriented requirements analysis

Thesis submitted to Faculdade de Ciências e Tecnologia of Universidade Nova de Lisboa in partial fulfilment of the requirements for the degree of Master in Computer Science

Pozzolanic components in lime mortars: correlating behaviour, composition and microstructure.

Int. J. for Restoration of Buildings and Monuments, vol.11, nº 2 (2005), p.111-118

Landscape composition and climatic parameters significant in the spread of an invasive species (Pine wood Nematode)

Dissertação para obtenção do Grau de Mestre em Engenharia do Ambiente

Malnutrição em idosos na admissão hospital actuação perante a sua elevado prevalência

RESUMO: Ao longo das últimas décadas a redistribuição etária da população mundial tem vindo a apresentar um aumento do número de pessoas com 65 ou mais anos, integrando um grupo populacional comummente designado por população idosa. Importa aprofundar mecanismos fisiológicos que conduzem ao envelhecimento e de que forma podem condicionar não só aspetos clínicos, como também nutricionais, entre outros, com a perspetiva da sua origem no aparecimento de doenças crónicas. Com esse enfoque, a desnutrição na pessoa idosa é hoje considerada pela European Nutrition for Health Alliance(ENHA) um problema de saúde pública. Está descrito que a sua prevalência ronda os 60% a nível de instituições hospitalares, 40% em unidades residenciais e 5 a 10% na pessoa idosa a residir em domicílio próprio ou de familiares, e na sua maioria permanece por diagnosticar e tratar. Assim, foi objetivo deste estudo caracterizar e estimar a prevalência da desnutrição e do risco de desnutrição na pessoa idosa, nas primeiras 72 horas de admissão hospitalar. Aplicou-se um estudo observacional, analítico, transversal, quantitativo e correlacional, cujos dados foram recolhidos por entrevista ao próprio e por observação. O estudo desenvolveu-se em duas vertentes de investigação, uma focada na caracterização da desnutrição em pessoas idosas institucionalizadas em hospitais portugueses da zona centro e sul do Continente e Madeira, nos períodos de julho/agosto de 2009, abril/junho de 2010, maio/julho de 2011, através do MNA®. A outra, uma avaliação nutricional detalhada, efetuada no Centro Hospitalar de Lisboa Central – Hospital de Santa Marta, EPE, entre o início de janeiro de 2009 e o fim de janeiro de 2010, sendo a amostra recrutada de entre os idosos de ambos os sexos, internados. Foram estudados dados sociodemográficos, de saúde e feita uma avaliação nutricional extensa. A avaliação nutricional constou de colheita de parâmetros laboratoriais (hematológicos e bioquímicos) e antropométricos (índice de massa corporal (IMC), prega cutânea tricipital(PCT), prega cutânea subescapular (PCSE), perímetro braquial (PB), adequação do perímetro braquial (APB), área muscular braquial (AMB) e perímetro Geminal (PG), análise da composição corporal (Massa Gorda Corporal (MGC), Massa Isenta de Gordura (MIG)),caracterização de um dia alimentar tipo e questionário Mini Nutritional Assessment Long Form®– MNA LF®. Dos dados obtidos em hospitais portugueses, destaca-se que dos 402 idosos avaliados, 53% eram do sexo masculino, tinham uma idade média de 75,8 + 6,52 (65 – 100) e segundo o MNA® 57,5% encontravam-se Desnutridos ou em Risco de Desnutrição.Na amostra, dos dados obtidos, a nível sociodemográfico salienta-se que 50% dos doentes eram do sexo masculino, a idade média rondava os 75,5 + 7,22 (65 – 100) anos, 55% eram naturais de Lisboa e 80% residiam em Lisboa e Vale do Tejo, 38% não tiveram estudos formais e 43% fizeram-no apenas até ao 4º ano de escolaridade. Em relação aos dados de saúde, a maioria dos doentes foi admitida através do Serviço de Urgência do Centro Hospitalar de Lisboa Central – Hospital de São José e foram internados no Serviço de Medicina (38%) e no Serviço de Cardiologia (30%), por patologia médica (38%) e patologia do sistema circulatório (56%). Nos hábitos de vida, quanto à mobilidade, um terço dos doentes estavam acamados e os restantes deambulavam ou tinham uma mobilidade normal, 74% não apresentaram hábitos etanólicos regulares, 19% apresentavam um consumo elevado (> 30g de etanol/dia); 95% dos doentes não apresentavam hábitos tabágicos. Relativamente à caracterização nutricional, os valores médios encontrados em relação aos parâmetros laboratoriais revelaram-se inferiores aos valores padrão para a idade e sexo e eram inferiores no sexo feminino. Na caracterização antropométrica verificaram-se os seguintes achados: o cálculo do IMC mostrou-se pouco sensível na identificação de doentes desnutridos; a PCT e a PCSE revelaram valores de massa gorda dentro do intervalo considerado normal;segundo o PB, 88% não apresentavam valor indicativo de desnutrição e 8% estavam desnutridos; a APB identificou 50% de doentes desnutridos; a AMB, revelou que 97% dos homens e 95% das mulheres apresentavam deficit da massa magra e segundo o PG, 18% apresentavam um valor inferior a 31cm descritor de desnutrição. Na análise da composição corporal verificou-se que ambos os sexos apresentavam uma percentagem de MGC classificada como demasiado alta e que esta era superior nas mulheres em relação aos homens. Ao analisar a ingestão nutricional verificou-se que esta era inferior às Dietary Reference Intakes (DRIs) para a ingestão hídrica (p=0,00), energética (p=0,00), proteica (p=0,00), lipídica (p=0,01), MUFA (p=0,00), PUFA (p=0,00), e glícidos (p=0,00), fibra (p=0,02), potássio (p=0,00), cálcio (p=0,00), magnésio (p=0,00), fósforo (p=0,00), zinco (p=0,00), vitamina D (p=0,00), vitamina E (p=0,00) e folato (p=0,00). No que diz respeito ao MNA®, a sua aplicação permitiu identificar 62% de situações de risco nutricional ou de desnutrição já instalada. Valores de MNA® indicativos de desnutrição ou risco estavam associados a níveis de escolaridade mais baixos (r=0,32; p=0,00). Verificou-se correlação entre o MNA® e a PCT (r=0,30;p=0,00), PCSE (r=0,19;p=0,03) e PG (r=0,27;p=0,00). Na análise da amostra por sexo e escalão etário, apenas se distinguiram as mulheres mais velhas, que apresentaram situação de IMC indicador de risco de desnutrição (IMC <23,5 + 2,9, (r=0,42;p=0,02)), e de valores médios de PB de 25,6+3,84cm (r=0,42;p=0,01), em situações de menor mobilidade caraterizados pelo MNA®. Os homens maisvelhos apresentaram correlação entre o MNA® e PCSE (r=0,41;p=0,02), APMB (r=0,57;p=0,00)e PG (r=0,55;p=0,00), e as mulheres mais velhas apenas com a PCT (r=0,39;p=0,02). A análise multivariada do MNA® em função do sexo e do escalão etário, revelou que estes são independentes. Os homens apresentaram valores médios de MNA® superiores às mulheres e à medida que a idade aumenta, os valores de MNA® em ambos os sexos diminuem, sendo indicativos de risco de desnutrição. Consideramos que, tendo em conta a natureza e objetivos do presente estudo, foi possível caracterizar e estimar a prevalência da desnutrição e do risco de desnutrição em pessoas idosas nas primeiras 72 horas de admissão hospitalar. Os resultados obtidos sinalizam a sua elevada prevalência e alertam para a necessidade de procedimentos protocolados de avaliação e intervenção nutricional da população idosa na admissão hospitalar. Para este efeito a aplicação do MNA® provou a sua aplicabilidade, assim como a medição e cálculo da AMB, que poderão ser muito precocemente aplicados e contribuir para potenciar melhorias do estado de saúde e diminuir o tempo de internamento, nomeadamente de pessoas idosas. Em relação ao padrão alimentar, este estudo contribuiu para uma chamada de atenção dos profissionais de saúde que a população idosa pode apresentar carências nutricionais na admissão, e que estas se não forem devidamente sinalizadas e colmatadas tendem a agravar-se durante o internamento podendo contribuir para o aumento da morbilidade.-------------ABSTRACT:Over the last decades the age redistribution group of the population worldwide has been presenting an increasing number of people aged 65 years or more, incorporating a population group commonly referred to as the elderly population. It´s important to further analyze the physiological mechanisms that lead to aging and how they might influence not only clinical aspects, but also nutritional, among others, with the perspective of their origin in the onset of chronic diseases. With this approach, malnutrition in the elderly is now considered by the European Nutrition for Health Alliance (ENHA) a public health problem. It is reported that its prevalence is around 60% at the level of hospital units, 40% in residential units and 5 to 10% in the elderly living in their own home or family's, and mostly remains to diagnose and treat. The aim of this study was to characterize and estimate the prevalence of malnutrition and risk of malnutrition in the elderly, in the first 72 hours of hospital admission. We applied an observational, analytical, cross-sectional and correlacional quantitative type of study and data were collected by interview and observation itself. The study was developed in two lines of research: one focused on the characterization of malnutrition in elderly institutionalized in Portuguese hospitals, in the central and southern mainland and Madeira, in the periods between July - August 2009, April - June 2010, May - July 2011, through the MNA®; and the other: a detailed nutritional assessment, conducted in Hospital Lisbon Center - Hospital de Santa Marta, EPE, between early January 2009 and late January 2010, and the sample was recruited from among the elderly of both sexes at hospital admission. We studied intensively sociodemographic, health and nutritional assessment done extensive. Nutritional evaluation consisted of harvesting different parameters: hematological, biochemical and anthropometric (body mass index (BMI), triceps skinfold (TSF), sub-scapular skinfold (SSF), arm circumference (AC), arm muscle area (AMA), geminal perimeter (GP), analysis of body composition (Fat Mass (FM), Fat Free Mass (FFM)), characterization of a daily food type and Mini Nutritional Assessment Long Form® questionnaire - MNA LF®. Form the data obtained in Portuguese hospitals, it is noteworthy that of the 402 patients included, 53% were male, had a mean age of 75,8 + 6,52 (65 - 100) and, according to the MNA®, 57,5% were malnourished or at risk of malnutrition. In the sample, from the sociodemographic data obtained, we saw that 50% of patients were male, the average age was around 75,5 + 7,22 years (65-100), 55% were from Lisbon and 80 %lived in Lisbon, 38% had no formal education and 43% did so only until the 4th grade. Regarding health data, the majorities of patients were admitted through the ER of Hospital Lisbon Center - S. José Hospital - and were admitted to the Medicine Unit (38%) and to the Cardiology Unit (30%), by medical pathology (38%) and circulatory system disease (56%). In regard to lifestyle, and considering mobility, one third of patients were bedridden and the rest were ambulating or had a normal mobility. 74% had no regular ethanol habits, 19% had a high intake (> 30 g ethanol / day); 95% of the patients had no smoking habits. Regarding nutritional assessment, the mean values for laboratory parameters proved inferior to standard values for age and sex and were lower in females. In anthropometric assessment these were the findings: BMI calculation showed to be scarcely sensitive in the identification of undernourished patients; the TSF and SSF revealed values of fat mass within the normal range; in AC, 88% did not have an indicative value of malnutrition and 8% were malnourished; in AMA, 97% of men and 95% women had a deficit of lean mass and in GP, 18% had a value of less than the 31cm malnutrition descriptor. In body composition analysis found that both sexes showed a percentage of FM ranked too high and this was higher in women compared to men. By analyzing the nutritional intake was found that this was less than the Dietary Reference Intakes (DRIs) for water intake (p=0,00), energy (p=0,00), protein (p=0,00), lipid (p=0,01), MUFA (p=0,00), PUFA (p=0,00), carbohydrates (p=0,00), fiber (p=0,02), potassium (p=0,00), calcium (p=0,00), magnesium (p=0,00), phosphorus (p=0,00), zinc (p=0,00), vitamin D (p=0,00), vitamin E (p=0,00) and folate (p=0,00). Regarding MNA®, its application identified 62% of cases of nutritional risk or malnutrition already installed. MNA® values indicative of malnutrition or risk were associated with lower levels of education (r=0,32; p=0,00). There was a correlation between the MNA ® and TSF (r =0,30, p = 0,00), SFF (r = 0,19, p = 0,03) and GP (r=0,27, p = 0,00). In the analysis of the sample by gender and age group, the highlight was in older women who had BMI status indicator of malnutrition risk (BMI <23,5 + 2,9 (r=0,42;p=0,02)) and mean values of AC 25,6 +3,84cm (r=0,42; p=0,01), in situations characterized by low mobility MNA®. Older men showed a correlation between the MNA® and SFF (r = 0,41; p = 0,02), AMA (r = 0,57; p = 0,00) and GP (r=0,55;p=0,00), and in older women only TSF showed a correlation(r = 0,39; p =0,02). Multivariate analysis of the MNA® by gender and age group, revealed that they are independent. The men had MNA® mean superior to women, and as the age increases, the values of MNA® in both sexes declined, being indicative of risk of malnutrition. We believe that, given the nature and objectives of the present study, it allowed us to characterize and estimate the prevalence of risk of malnutrition and malnutrition in older people during the first 72 hours of hospital admission. The results indicate a high prevalence and point to the need for protocol procedures of nutritional assessment and intervention in the elderly population at hospital admission. For this purpose the application of MNA® has proved its applicability, as well as measuring and calculating AMA, which may be applied in early stages thus contributing to enhance health state improvements and to shorten the time of hospitalization, particularly in elderly people. In relation to dietary pattern, this study contributed to call of attention from health professionals that the elderly may have nutritional deficiencies on admission, and that these are not properly marked and addressed tend to worsen during hospitalization may contribute to increased morbidity.

Storage of water molecules into biomimetic heterostructures: the role of roughness

The development of devices based on heterostructured thin films of biomolecules conveys a huge contribution on biomedical field. However, to achieve high efficiency of these devices, the storage of water molecules into these heterostructures, in order to maintain the biological molecules hydrated, is mandatory. Such hydrated environment may be achieved with lipids molecules which have the ability to rearrange spontaneously into vesicles creating a stable barrier between two aqueous compartments. Yet it is necessary to find conditions that lead to the immobilization of whole vesicles on the heterostructures. In this work, the conditions that govern the deposition of open and closed liposomes of 1.2-dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (sodium Salt) (DPPG) onto polyelectrolytes cushions prepared by the layer-by-layer (LbL) method were analyzed. Electronic transitions of DPPG molecules as well as absorption coefficients were obtained by vacuum ultraviolet spectroscopy, while the elemental composition of the heterostructures was characterized by x-ray photoelectron spectroscopy (XPS). The presence of water molecules in the films was inferred by XPS and infrared spectroscopy. Quartz crystal microbalance (QCM) data analysis allowed to conclude that, in certain cases, the DPPG adsorbed amount is dependent of the bilayers number already adsorbed. Moreover, the adsorption kinetics curves of both adsorbed amount and surface roughness allowed to determine the kinetics parameters that are related with adsorption processes namely, electrostatic forces, liposomes diffusion and lipids re-organization on surface. Scaling exponents attained from atomic force microscopy images statistical analysis demonstrate that DPPG vesicles adsorption mechanism is ruled by the diffusion Villain model confirming that adsorption is governed by electrostatic forces. The power spectral density treatment enabled a thorough description of the accessible surface of the samples as well as of its inner structural properties. These outcomes proved that surface roughness influences the adsorption of DPPG liposomes onto surfaces covered by a polyelectrolyte layer. Thus, low roughness was shown to induce liposome rupture creating a lipid bilayer while high roughness allows the adsorption of whole liposomes. In addition, the fraction of open liposomes calculated from the normalized maximum adsorbed amounts decreases with the cushion roughness increase, allowing us to conclude that the surface roughness is a crucial variable that governs the adsorption of open or whole liposomes. This conclusion is fundamental for the development of well-designed sensors based on functional biomolecules incorporated in liposomes. Indeed, LbL films composed of polyelectrolytes and liposomes with and without melanin encapsulated were successfully applied to sensors of olive oil.

Characterization of molecular damage induced by UV photons and carbon ions on biomimetic heterostructures

The study of the effect of radiation on living tissues is a rather complex task to address mainly because they are made of a set of complex functional biological structures and interfaces. Particularly if one is looking for where damage is taking place in a first stage and what are the underlying reaction mechanisms. In this work a new approach is addressed to study the effect of radiation by making use of well identified molecular hetero-structures samples which mimic the biological environment. These were obtained by assembling onto a solid support deoxyribonucleic acid (DNA) and phospholipids together with a soft water-containing polyelectrolyte precursor in layered structures and by producing lipid layers at liquid/air interface with DNA as subphase. The effects of both ultraviolet (UV) radiation and carbon ions beams were systematically investigated in these heterostructures, namely damage on DNA by means vacuum ultraviolet (VUV), infrared (IR), X-Ray Photoelectron (XPS) and impedance spectroscopy. Experimental results revealed that UV affects furanose, PO2-, thymines, cytosines and adenines groups. The XPS spectrometry carried out on the samples allowed validate the VUV and IR results and to conclude that ionized phosphate groups, surrounded by the sodium counterions, congregate hydration water molecules which play a role of UV protection. The ac electrical conductivity measurements revealed that the DNA electrical conduction is arising from DNA chain electron hopping between base-pairs and phosphate groups, with the hopping distance equal to the distance between DNA base-pairs and is strongly dependent on UV radiation exposure, due loss of phosphate groups. Characterization of DNA samples exposed to a 4 keV C3+ ions beam revealed also carbon-oxygen bonds break, phosphate groups damage and formation of new species. Results from radiation induced damage carried out on biomimetic heterostructures having different compositions revealed that damage is dependent on sample composition, with respect to functional targeted groups and extent of damage. Conversely, LbL films of 1,2-dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt) (DPPG) liposomes, alternated with poly(allylamine hydrochloride) (PAH) revealed to be unaffected, even by prolonged UV irradiation exposure, in the absence of water molecules. However, DPPG molecules were damaged by the UV radiation in presence of water with cleavage of C-O, C=O and –PO2- bonds. Finally, the study of DNA interaction with the ionic lipids at liquid/air interfaces revealed that electrical charge of the lipid influences the interaction of phospholipid with DNA. In the presence of DNA in the subphase, the effects from UV irrladiation were seen to be smaller, which means that ionic products from biomolecules degradation stabilize the intact DPPG molecules. This mechanism may explain why UV irradiation does not cause immediate cell collapse, thus providing time for the cellular machinery to repair elements damaged by UV.

Analysis of metabolic flux distributions in relation to the extracellular environment in Avian cells

Continuous cell lines that proliferate in chemically defined and simple media have been highly regarded as suitable alternatives for vaccine production. One such cell line is the AG1.CR.pIX avian cell line developed by PROBIOGEN. This cell line can be cultivated in a fully scalable suspension culture and adapted to grow in chemically defined, calf serum free, medium [1]–[5]. The medium composition and cultivation strategy are important factors for reaching high virus titers. In this project, a series of computational methods was used to simulate the cell’s response to different environments. The study is based on the metabolic model of the central metabolism proposed in [1]. In a first step, Metabolic Flux Analysis (MFA) was used along with measured uptake and secretion fluxes to estimate intracellular flux values. The network and data were found to be consistent. In a second step, Flux Balance Analysis (FBA) was performed to access the cell’s biological objective. The objective that resulted in the best predicted results fit to the experimental data was the minimization of oxidative phosphorylation. Employing this objective, in the next step Flux Variability Analysis (FVA) was used to characterize the flux solution space. Furthermore, various scenarios, where a reaction deletion (elimination of the compound from the media) was simulated, were performed and the flux solution space for each scenario was calculated. Growth restrictions caused by essential and non-essential amino acids were accurately predicted. Fluxes related to the essential amino acids uptake and catabolism, the lipid synthesis and ATP production via TCA were found to be essential to exponential growth. Finally, the data gathered during the previous steps were analyzed using principal component analysis (PCA), in order to assess potential changes in the physiological state of the cell. Three metabolic states were found, which correspond to zero, partial and maximum biomass growth rate. Elimination of non-essential amino acids or pyruvate from the media showed no impact on the cell’s assumed normal metabolic state.

Choosing the gender and skill of politicians: List composition and electability across Brazilian municipalities

This work project studies the effect of variations in the proportion of female candidates on the quality of politicians. This effect was divided between nominated and elected body. Cross-sectional data was used for two elections, and an OLS as an IV approach. Results show that the existence of female candidates on parties’ list increases the quality of the nominated body. Moreover, contrary to what many advocate, increasing the presence of female candidates either increases or has no effect on the quality of the elected body. Results that were confirmed for the overall data and controlling for region factors only.

The role of s-nitrosothiols and rho-kinase 1 in glucose and lipid metabolism

RESUMO: Na sociedade contemporânea a diabetes tipo 2 e a obesidade estão a aumentar exponencialmente, representando um grave problema de saúde pública. De acordo com a IDF “A diabetes e a obesidade são o principal problema de saúde pública do século XXI’. Para além destas duas patologias, a prevalência de esteatose hepática não-alcoólica (NAFLD), entre a população obesa e diabética, é de cerca de 90%. O aumento da obesidade, diabetes e NAFLD tem uma forte correlação com o aumento do consumo de gorduras e açúcares, acompanhado de um decréscimo acentuado da actividade física. A obesidade, diabetes e NAFLD tem sido escrupolosamente investigada mas as terapêuticas disponíveis continuam a ser muito limitadas. Tendo em conta o número crescente e alarmante de obesos e diabéticos o conhecimento detalhado da patofisiologia da obesidade, diabetes e NAFLD, tendo em vista a necessidade extrema de desenvolvimento de novas estratégias terapêuticas, é da mais elevada urgência. O fígado é reconhecido como um orgão primordial no controlo da homeostase. No estado pós-prandial, o fígado converte a glucose em glicogénio e lípidos. Em contraste, no estado de jejum, o fígado promove a produção de glucose. Sistemas neuronais e hormonais, bem como o estado metabólico do fígado, controlam de forma muito precisa a alternância entre os diferentes substratos metabólicos, dependente do estado prandial. A insulina tem um papel central no controlo do metabolismo energético no fígado; se, por um lado, inibe a produção hepática de glucose e corpos cetónicos, por outro, promove a glicólise e a lipogénese. O metabolismo energético no fígado é também regulado por vários factores de transcrição e co-reguladores que, por sua vez, são regulados pela insulina, glucagina e outras hormonas metabólicas. Em conjunto, todos estes factores e reguladores vão controlar de forma muito estreita a gluconeogénese, a β-oxidação e a lipogénese, no fígado. Para além dos já conhecidos reguladores do metabolismo hepático, novas moléculas têm sido estudadas como tendo um papel fundamental na regulação do metabolismo energético no fígado. Qualquer desequilíbrio no metabolismo hepático vai contribuir para a insulino-resistência, NAFLD e diabetes tipo 2. O principal objectivo do trabalho de investigação aqui apresentado é o contributo para o estudo detalhado da patogénese da diabetes e obesidade, num contexto de dietas ricas em açúcares e gorduras, e com a perspectiva de explorar novas estratégias terapêuticas. Os objectivos específicos deste trabalho eram: primeiro, determinar se o tratamento com glutationo (GSH) e óxido nítrico (NO) era suficiente para melhorar a insulino-resistência associada ao elevado consumo de sacarose; segundo, determinar o papel da Rho-kinase 1 (ROCK1) na regulação do metabolismo hepático da glucose e dos lípidos; e terceiro, estudar o efeito do metilsulfonilmetano (MSM) em doenças metabólicas associadas à obesidade. Na primeira parte deste trabalho de investigação foram utilizados ratos Wistar machos sujeitos a uma dieta rica em sacarose (HS). Tal como esperado, estes animais apresentavam insulino-resistência e hiperinsulinémia. A dieta HS levou ao aumento dos níveis hepáticos de NO e ao decréscimo dos níveis de GSH no fígado. Em jejum, a administração intraportal de GSH e NO, a animais saudáveis promoveu um aumento significativo da sensibilidade à insulina. Também nestes animais, a administração intravenosa de S-nitrosotióis, compostos orgânicos que contém um grupo nitroso acoplado a um átomo de enxofre de um tiol, promoveu o aumento significativo da sensibilidade à insulina. Pelo contrário, em animais sujeitos à dieta HS, as doses padrão de GSH + NO e de S-nitrosotióis não conseguiram promover o aumento da sensibilidade à insulina. No entanto, ao aumentar a dose de S-nitrosotióis administrados por via intravenosa, foi possível observar o aumento da sensibilidade à insulina dependente da dose, indicando um possível papel dos S-nitrosotióis como sensibilizadores de insulina. O estudo detalhado do papel dos S-nitrosotióis na via de sinalização da insulina revelou que há um aumento da fosforilação do receptor da insulina (IR) e da proteína cinase B (Akt), sugerindo um efeito dos S-nitrosotióis nesta via de sinalização. Os resultados apresentados nesta primeira parte sugerem que os S-nitrosotióis promovem a correcta acção da insulina, podendo vir a ser importantes alvos terapêuticos. Na segunda parte deste trabalho de investigação utilizámos murganhos, com uma delecção específica da ROCK1 no fígado, e sujeitos a uma dieta rica em lípidos (HFD). Foi possível concluir que a ausência da ROCK1 no fígado previne a obesidade, melhora a sensibilidade à insulina e protege contra a esteatose hepática. A ausência de ROCK1 no fígado levou a um decréscimo significativo da expressão génica de genes associados à lipogénese, com uma diminuição acentuada do fluxo metabólico associado a esta via. Pelo contrário, a sobreexpressão de ROCK1, exclusivamente no fígado, promove a insulino-resistência e a esteatose hepática no contexto de obesidade induzida pela dieta. Para além disto, a delecção da ROCK1 no fígado de animais obesos e diabéticos, os murganhos deficientes em leptina, corroborou os dados obtidos no primeiro modelo animal, com a franca melhoria da hiperglicémia, hiperinsulinémia e esteatose hepática. Os dados que compõem esta parte do trabalho de investigação sugerem que a ROCK1 tem um papel crucial na regulação do metabolismo lipídico. Na terceira e última parte deste trabalho de investigação foi investigado o efeito do composto metilsulfunilmetano (MSM), um composto organosulfúrico naturalmente presente em plantas e utilizado também como suplemento dietético, em murganhos obesos e insulino-resistentes, por exposição a uma dieta rica em lípidos (DIO). O tratamento com MSM melhorou a insulino-resistência e protegeu contra a esteatose hepática. O conteúdo hepático em triglicéridos e colesterol também diminuíu de forma significativa nos animais DIO sujeitos ao tratamento com MSM, bem como a expressão génica associada à lipogénese. Para além disto, o tratamento com MSM levou a uma diminuição da expressão génica associada à inflamação. De realçar que o tratamento com MSM levou a uma melhoria do perfil hematopoiético destes animais, tanto na medula óssea como no sangue. Para comprovar o efeito benéfico do MSM na obesidade e insulino-resistência utilizámos murganhos deficientes no receptor da leptina, e por isso obesos e diabéticos, tendo observado um perfil semelhante ao obtido para murganhos sujeitos a uma dieta rica em lípidos e tratados com MSM. Concluímos, através dos dados recolhidos, que o MSM como suplemento pode ter efeitos benéficos na hiperinsulinémia, insulino-resistência e inflamação que caracterizam a diabetes tipo 2. Em resumo, os dados obtidos neste trabalho de investigação mostram que os S-nitrosotióis podem ter um papel importante como sensibilizadores da insulina, promovendo um aumento da sensibilidade à insulina num contexto de dietas ricas em sacarose. Para além disto, estudos in vitro, sugerem que os S-nitrosotióis regulam, especificamente, a via de sinalização da insulina. Este trabalho teve também como objectivo o estudo da ROCK1 como regulador do metabolismo da glucose e dos lípidos no fígado. Através do estudo de animais com uma delecção ou uma sobreexpressão da ROCK1 no fígado mostrou-se que esta tem um papel crucial na patogénese da obesidade e diabetes tipo 2, especificamente através do controlo da lipogénese de novo. Finalmente, foi também objectivo deste trabalho, explorar o efeito do MSM em animais DIO e deficientes em leptina. O tratamento com MSM protege de forma evidente contra a obesidade e insulino-resistência, com especial enfâse para a capacidade que esta molécula demonstrou ter na protecção contra a inflamação. Em conjunto os vários estudos aqui apresentados mostram que tanto os S-nitrosotióis como a ROCK1 têm um papel na patogénese da obesidade e diabetes tipo 2 e que a utilização de MSM como suplemento às terapêuticas convencionais pode ter um papel no tratamentos de doenças metabólicas.-------------------------------ABSTRACT: In modern western societies type 2 diabetes and obesity are increasing exponentially, representing a somber public concern. According to the International Diabetes Federation (IDF) ‘Diabetes and Obesity are the biggest public health challenges of the 21st century’. Aside from these the prevalence of nonalcoholic fatty liver disease (NAFLD), among the diabetic and obese population, is as high as 90%. It is now well established that the increase in obesity, diabetes and NAFLD strongly correlates with an increase in fat and sugar intake in our diet, alongside physical inactivity. The pathogenesis of obesity, diabetes and NAFLD has been thoroughly studied but the treatment options available are still narrow. Considering the alarming number in the obese and diabetic population the complete understanding of the pathogenesis, keeping in mind that new therapeutic strategies need to be attained, is of the highest urgency. The liver has been well established as a fundamental organ in regulating whole-body homeostasis. In the fed state the liver converts the glucose into glycogen and lipids. Conversely, in the fasted state, glucose will be produced in the liver. Neuronal and hormonal systems, as well as the hepatic metabolic states, tightly control the fast to fed switch in metabolic fuels. Insulin has a central role in controlling hepatic energy metabolism, by suppressing glucose production and ketogenesis, while stimulating glycolysis and lipogenesis. Liver energy metabolism is also regulated by various transcription factors and coregulators that are, in turn, regulated by insulin, glucagon and other metabolic hormones. Together, these regulators will act to control gluconeogenesis, β-oxidation and lipogenesis in the liver. Aside from the well-established regulators of liver energy metabolism new molecules are being studied has having a role in regulating hepatic metabolism. Any imbalance in the liver energy metabolism is a major contributor to insulin resistance, NAFLD and type 2 diabetes. The overall goal of this research work was to contribute to the understanding of the pathogenesis of diabetes and obesity, on a setting of high-sucrose and high-fat diets, and to explore potential therapeutic options. The specific aims were: first, to determine if treatment with glutathione (GSH) and nitric oxide (NO) was sufficient to ameliorate insulin resistance induced by high-sucrose feeding; second, to determine the physiological role of rho-kinase 1 (ROCK1) in regulating hepatic and lipid metabolism; and third, to study the effect of methylsulfonylmethane (MSM) on obesity-linked metabolic disorders. In the first part of this research work we used male Wistar rats fed a high-sucrose (HS) diet. As expected, rats fed a HS diet were insulin resistant and hyperinsulinemic. HS feeding increased hepatic levels of NO, while decreasing GSH. In fasted healthy animals administration of both GSH and NO, to the liver, was able to increase insulin sensitivity. Intravenous administration of S-nitrosothiols, organic compounds containing a nitroso group attached to the sulfur atom of a thiol, in fasted control animals also increased insulin sensitivity. Under HS feeding the standard doses of GSH + NO and S-nitrosothiols were unable to promote an increase in insulin sensitivity. However, the intravenous administration of increasing concentrations of S-nitrosothiols was able to restore insulin sensitivity, suggesting that S-nitrosothiols have an insulin sensitizing effect. Investigation of the effect of S-nitrosothiols on the insulin signaling pathway showed increased phosphorylation of the insulin receptor (IR) and protein kinase B (Akt), suggesting that S-nitrosothiols may have an effect on the insulin signaling pathway. Together, these data showed that S-nitrosothiols promote normal insulin action, suggesting that they may act as potential pharmacological tools. In the second part of this research work we used liver-specific ROCK1 knockout mice fed a high-fat (HF) diet. Liver-specific deletion of ROCK1 prevented obesity, improved insulin sensitivity and protected against hepatic steatosis. Deficiency of ROCK1 in the liver caused a significant decrease in the gene expression of lipogenesis associated gene, ultimately leading to decreased lipogenesis. Contrariwise, ROCK1 overexpression in the liver promoted insulin resistance and hepatic steatosis in diet-induced obesity. Furthermore, liver-specific deletion of ROCK1 in obese and diabetic mice, the leptin-deficient mice, improved the typical hyperglycemia, hyperinsulinemia and liver steatosis. Together, these data identify ROCK1 as a crucial regulator of lipid metabolism. In the third and final part of this research work we investigated the effect of MSM, an organosulfur compound naturally found in plants and used as a dietary supplement, on diet-induced obese (DIO) and insulin resistant mice. MSM treatment ameliorated insulin resistance and protected against hepatosteatosis. Hepatic content in triglycerides and cholesterol was significantly decreased by MSM treatment, as well as lipogenesis associated gene expression. Furthermore, MSM treated mice had decreased inflammation associated gene expression in the liver. Importantly, FACS analysis showed that MSM treatment rescued the inflammatory hematopoietic phenotype of DIO mice in the bone marrow and the peripheral blood. Moreover, MSM treatment of the obese and diabetic mice, the leptin-deficient mice, resulted in similar effects as the ones observed for DIO mice. Collectively, these data suggest that MSM supplementation has a beneficial effect on hyperinsulinemia, insulin resistance and inflammation, which are often found in type 2 diabetes. In conclusion, this research work showed that S-nitrosothiols may play a role as insulin sensitizers, restoring insulin sensitivity in a setting of high-sucrose induced insulin resistance. Furthermore, in vitro studies suggest that S-nitrosothiols specifically regulate the insulin signaling pathway. This research work also investigated the role of hepatic ROCK1 in regulation of glucose and lipid metabolism. Using liver-specific ROCK 1 knockout and ROCK1 overexpressing mice it was shown that ROCK1 plays a role in the pathogenesis of obesity and type 2 diabetes, specifically through regulation of the de novo lipogenesis pathway. Finally, this research work aimed to explore the effect of MSM in DIO and leptin receptor-deficient mice. MSM strongly protects against obesity and insulin resistance, moreover showed a robust ability to decrease inflammation. Together, the individual studies that compose this dissertation showed that S-nitrosothiols and ROCK1 play a role in the pathogenesis of obesity and type 2 diabetes and that MSM supplementation may have a role in the treatment of metabolic disorders.

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