A Doença Celíaca

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Novos sistemas farmacêuticos para administração pulmonar

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Novos sistemas terapêuticos para administração nasal de fármacos

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

B Cell Activation in Insulin Resistance and Obesity

Our group has demonstrated that inflammatory diseases such as type 2 diabetes (DM), inflammatory bowel disease (IBD), and periodontal disease (PD) are associated with altered B cell function that may contribute to disease pathogenesis. B cells were found to be highly activated with characteristics of inflammatory cells. Obesity is a pre-disease state for cardiovascular disease and type 2 diabetes and is considered a state of chronic inflammation. Therefore, we sought to better characterize B cell function and phenotype in obese patients. We demonstrate that (Toll-like receptor) TLR4 and CD36 expression by B cells is elevated in obese subjects, suggesting increased sensing of lipopolysaccharide (LPS) and other TLR ligands. These ligands may be of microbial, from translocation from a leaky gut, or host origin. To better assess microbial ligand burden and host response in the bloodstream, we measured LPS binding protein (LBP), bacterial/permeability increasing protein (BPI), and high mobility group box 1 (HMGB1). Thus far, our data demonstrate an increase in LBP in DM and obesity indicating increased responses to TLR ligands in the blood. Interestingly, B cells responded to certain types of LPS by phosphorylating extracellular-signal-regulated kinases (ERK) 1/2. A better understanding of the immunological state of obesity and the microbial and endogenous TLR ligands that may be activating B cells will help identify novel therapeutics to reduce the risk of more dangerous conditions, such as cardiovascular disease.

Plasmid biology of natural Lactococcus lactis strains and molecular mechanisms of bacteriophage-host interaction

Lacticin 3147, enterocin AS-48, lacticin 481, variacin, and sakacin P are bacteriocins offering promising perspectives in terms of preservation and shelf-life extension of food products and should find commercial application in the near future. The studies detailing their characterization and bio-preservative applications are reviewed. Transcriptomic analyses showed a cell wall-targeted response of Lactococcus lactis IL1403 during the early stages of infection with the lytic bacteriophage c2, which is probably orchestrated by a number of membrane stress proteins and involves D-alanylation of membrane lipoteichoic acids, restoration of the physiological proton motive force disrupted following bacteriophage infection, and energy conservation. Sequencing of the eight plasmids of L. lactis subsp. cremoris DPC3758 from raw milk cheese revealed three anti-phage restriction/modification (R/M) systems, immunity/resistance to nisin, lacticin 481, cadmium and copper, and six conjugative/mobilization regions. A food-grade derivative strain with enhanced bacteriophage resistance was generated via stacking of R/M plasmids. Sequencing and functional analysis of the four plasmids of L. lactis subsp. lactis biovar. diacetylactis DPC3901 from raw milk cheese revealed genes novel to Lactococcus and typical of bacteria associated with plants, in addition to genes associated with plant-derived lactococcal strains. The functionality of a novel high-affinity regulated system for cobalt uptake was demonstrated. The bacteriophage resistant and bacteriocin-producing plasmid pMRC01 places a metabolic burden on lactococcal hosts resulting in lowered growth rates and increased cell permeability and autolysis. The magnitude of these effects is strain dependent but not related to bacteriocin production. Starters’ acidification capacity is not significantly affected. Transcriptomic analyses showed that pMRC01 abortive infection (Abi) system is probably subjected to a complex regulatory control by Rgg-like ORF51 and CopG-like ORF58 proteins. These regulators are suggested to modulate the activity of the putative Abi effectors ORF50 and ORF49 exhibiting topology and functional similarities to the Rex system aborting bacteriophage λ lytic growth.

Role of mast cells in inflammatory bowel disease and inflammation-associated colorectal neoplasia in IL-10-deficient mice.

BACKGROUND: Inflammatory bowel disease (IBD) is hypothesized to result from stimulation of immune responses against resident intestinal bacteria within a genetically susceptible host. Mast cells may play a critical role in IBD pathogenesis, since they are typically located just beneath the intestinal mucosal barrier and can be activated by bacterial antigens. METHODOLOGY/PRINCIPAL FINDINGS: This study investigated effects of mast cells on inflammation and associated neoplasia in IBD-susceptible interleukin (IL)-10-deficient mice with and without mast cells. IL-10-deficient mast cells produced more pro-inflammatory cytokines in vitro both constitutively and when triggered, compared with wild type mast cells. However despite this enhanced in vitro response, mast cell-sufficient Il10(-/-) mice actually had decreased cecal expression of tumor necrosis factor (TNF) and interferon (IFN)-gamma mRNA, suggesting that mast cells regulate inflammation in vivo. Mast cell deficiency predisposed Il10(-/-) mice to the development of spontaneous colitis and resulted in increased intestinal permeability in vivo that preceded the development of colon inflammation. However, mast cell deficiency did not affect the severity of IBD triggered by non-steroidal anti-inflammatory agents (NSAID) exposure or helicobacter infection that also affect intestinal permeability. CONCLUSIONS/SIGNIFICANCE: Mast cells thus appear to have a primarily protective role within the colonic microenvironment by enhancing the efficacy of the mucosal barrier. In addition, although mast cells were previously implicated in progression of sporadic colon cancers, mast cells did not affect the incidence or severity of colonic neoplasia in this inflammation-associated model.

Metamaterial-enhanced coupling between magnetic dipoles for efficient wireless power transfer

Nonradiative coupling between conductive coils is a candidate mechanism for wireless energy transfer applications. In this paper we propose a power relay system based on a near-field metamaterial superlens and present a thorough theoretical analysis of this system. We use time-harmonic circuit formalism to describe all interactions between two coils attached to external circuits and a slab of anisotropic medium with homogeneous permittivity and permeability. The fields of the coils are found in the point-dipole approximation using Sommerfeld integrals which are reduced to standard special functions in the long-wavelength limit. We show that, even with a realistic magnetic loss tangent of order 0.1, the power transfer efficiency with the slab can be an order of magnitude greater than free-space efficiency when the load resistance exceeds a certain threshold value. We also find that the volume occupied by the metamaterial between the coils can be greatly compressed by employing magnetic permeability with a large anisotropy ratio. © 2011 American Physical Society.

Inhibition of beta-adrenergic receptor kinase prevents rapid homologous desensitization of beta 2-adrenergic receptors.

Homologous (agonist-specific) desensitization of beta-adrenergic receptors (beta ARs) is accompanied by and appears to require phosphorylation of the receptors. We have recently described a novel protein kinase, beta AR kinase, which phosphorylates beta ARs in vitro in an agonist-dependent manner. This kinase is inhibited by two classes of compounds, polyanions and synthetic peptides derived from the beta 2-adrenergic receptor (beta 2AR). In this report we describe the effects of these inhibitors on the process of homologous desensitization induced by the beta-adrenergic agonist isoproterenol. Permeabilization of human epidermoid carcinoma A431 cells with digitonin was used to permit access of the charged inhibitors to the cytosol; this procedure did not interfere with the pattern of isoproterenol-induced homologous desensitization of beta 2AR-stimulated adenylyl cyclase. Inhibitors of beta AR kinase markedly inhibited homologous desensitization of beta 2ARs in the permeabilized cells. Inhibition of desensitization by heparin, the most potent of the polyanion inhibitors of beta AR kinase, occurred over the same concentration range (5-50 nM) as inhibition of purified beta AR kinase assessed in a reconstituted system. Inhibition of desensitization by heparin was accompanied by a marked reduction of receptor phosphorylation in the permeabilized cells. Whereas inhibitors of beta AR kinase inhibited homologous desensitization, inhibitors of protein kinase C and of cyclic-nucleotide-dependent protein kinases were ineffective. These data establish that phosphorylation of beta ARs by beta AR kinase is an essential step in homologous desensitization of the receptors. They further suggest a potential therapeutic value of inhibitors of beta AR kinase in inhibiting agonist-induced desensitization.

Low Molecular Weight Opioid Peptide Esters Could be Developed as a New Class of Analgesics.

Low molecular weight opioid peptide esters (OPE) could become a class of analgesics with different side effect profiles than current opiates. OPE may have sufficient plasma stability to cross the blood brain barrier (BBB), undergo ester hydrolysis and produce analgesia. OPE of dipeptides, tyr-pro and tyr-gly conjugated to ethanol have a structure similar to the anesthestic agent, etomidate. Based upon the analgesic activity of dipeptide opioids, Lipinski's criteria, and permeability of select GABA esters to cross the BBB, opioid peptides (OP) conjugated to ethanol, cholesterol or 3-glucose are lead recommendations. Preliminary animal data suggests that tyr-pro-ethyl ester crosses the BBB and unexpectedly produces hyperalgesia. Currently, there are no approved OP analgesics available for clinical use. Clinical trials of good manufacturing practice OP administered to patients suffering from chronic pain with indwelling intrathecal pumps could resolve the issue that OP may be superior to opiates and may redirect research.

High-Resolution CT for Small-Animal Imaging Research

Preclinical imaging has a critical role in phenotyping, in drug discovery, and in providing a basic understanding of mechanisms of disease. Translating imaging methods from humans to small animals is not an easy task. The purpose of this work is to review high-resolution computed tomography (CT) also known as micro-CT for small-animal imaging. We present the principles, the technologies, the image quality parameters, and the types of applications. We show that micro-CT can be used to provide not only morphological but also functional information such as cardiac function or vascular permeability. Another way in which micro-CT can be used in the study of both function and anatomy is by combining it with other imaging modalities, such as positron emission tomography or single-photon emission tomography. Compared to other modalities, micro-CT imaging is usually regarded as being able to provide higher throughput at lower cost and higher resolution. The limitations are usually associated with the relatively poor contrast mechanisms and the radiation damage, although the use of novel nanoparticle-based contrast agents and careful design of studies can address these limitations.

A comprehensive copper stockpile leach model: background and parameter sensitivity

The design and development of a comprehensive computational model of a copper stockpile leach process is summarized. The computational fluid dynamic software framework PHYSICA+ and various phenomena were used to model transport phenomena, mineral reaction kinetics, bacterial effects, and heat, energy and acid balances for the overall leach process. In this paper, the performance of the model is investigated, in particular its sensitvity to particle size and ore permeability. A combination of literature and laboratory sources was used to parameterize the model. The simulation results from the leach model are compared with closely controlled column pilot scale tests. The main performance characteristics (e.g. copper recovery rate) predicted by the model compare reasonably well with the experimental data and clearly reflect the qualitiative behavior of the process in many respects. The model is used to provide a measure of the sensitivity of ore permeability on leach behavior, and simulation results are examined for several different particle size distributions.

Observations and analysis of a 63-year-old reinforced concrete promenade railing exposed to the North Sea

Despite being exposed to the harsh sea-spray environment of the North Sea at Arbroath, Scotland, for over 63 years, many of the reinforced concrete precast beam elements of the 1.5 km long promenade railing are still in very good condition and show little evidence of reinforcement corrosion. In contrast, railing replacements constructed in about 1968 and in 1993 are almost all badly cracked as a result of extensive corrosion of the longitudinal reinforcement. This is despite the newer concrete appearing to be of better quality than the 1943 concrete. Statistics for maximum crack width for each of the three populations, based on measurements made in 2004 and in 2006, are presented. In situ and laboratory measurements show that the 1943 concrete appears to have high permeability but it also shows high electrical resistivity. Chloride penetration measurements show the 1943 and 1993 concretes to have similar chloride profiles and similar chloride concentrations at the reinforcement bars. This is inconsistent with the 1943 beams showing much less reinforcement corrosion than their later replacements and casts doubt on the conventional practice for durability design focusing on reducing concrete permeability through denser concretes or greater cover.

The impact of powder flowing properties on dosator filling system operation

Purpose: To study the impact of powder flow properties on dosator filling systems, with particular focus on improvements in dose weight accuracy and repeatability. Method: This study evaluates a range of critical powder flow properties such as: flow function, cohesion, wall friction, adhesion to wall surfaces, density/compressibility data, stress ratio “K” and gas permeability. The characterisations of the powders considered in this study were undertaken using an annular shear cell using a sample size of 0.5 litres. This tester also incorporated the facility to measure bed expansion during shear in addition to contraction under consolidation forces. A modified Jenike type linear wall friction tester was used to develop the failure loci for the powder sample in conjunction with multiple wall samples (representing a variety of material types and surface finishes). Measurements of the ratio of applied normal stress versus lateral stress were determined using a piece of test equipment specifically designed for the purpose. Results: The correct characterisation of powders and the incorporation of this data into the design of process equipment are recognised as critical for reliable and accurate operation. An example of one aspect of this work is the stress ratio “K”. This characteristic is not well understood or correctly interpreted in many cases – despite its importance. Fig 1 [Omitted] (illustrates a sample of test data. The slope of the line gives the stress ratio in a uniaxial compaction system – indicating the behaviour of the material under compaction during dosing processes. Conclusions: A correct assessment of the bulk powder properties for a given formulation can allow prediction of: cavity filling behaviour (and hence dosage), efficiency of release from dosator, and strength and stability of extruded dose en route to capsule filling Influences over the effectiveness of dosator systems have been shown to be impacted upon by: bed pre-compaction history, gas permeability in the bed (with respect to local density effects), and friction effects for materials of construction for dosators

Modelling of dosator operation for improved manufacturing performance

Purpose: To develop an improved mathematical model for the prediction of dose accuracy of Dosators - based upon the geometry of the machine in conjunction with measured flow properties of the powder. Methods: A mathematical model has been created, based on a analytical method of differential slices - incorporating measured flow properties. The key flow properties of interest in this investigation were: flow function, effective angle of wall friction, wall adhesion, bulk density, stress ratio K and permeability. To simulate the real process and (very importantly) validate the model, a Dosator test-rig has been used to measure the forces acting on the Dosator during the filling stage, the force required to eject the dose and the dose weight. Results: Preliminary results were obtained from the Dosator test-rig. Figure 1 [Omitted] shows the dose weight for different depths to the bottom of the powder bed at the end of the stroke and different levels of pre-compaction of the powder bed. A strong influence over dose weight arising from the proximity between the Dosator and the bottom of the powder bed at the end of the stroke and the conditions of the powder bed has been established. Conclusions: The model will provide a useful tool to predict dosing accuracy and, thus, optimise the future design of Dosator based equipment technology – based on measured bulk properties of the powder to be handled. Another important factor (with a significant influence) on Dosator processes, is the condition of the powder bed and the clearance between the Dosator and the bottom of the powder bed.

Borehole Optimisation System (BOS) - a case study assessing options for abstraction of urban groundwater in Nottingham, UK

The recognition that urban groundwater is a potentially valuable resource for potable and industrial uses due to growing pressures on perceived less polluted rural groundwater has led to a requirement to assess the groundwater contamination risk in urban areas from industrial contaminants such as chlorinated solvents. The development of a probabilistic risk based management tool that predicts groundwater quality at potential new urban boreholes is beneficial in determining the best sites for future resource development. The Borehole Optimisation System (BOS) is a custom Geographic Information System (GIs) application that has been developed with the objective of identifying the optimum locations for new abstraction boreholes. BOS can be applied to any aquifer subject to variable contamination risk. The system is described in more detail by Tait et al. [Tait, N.G., Davison, J.J., Whittaker, J.J., Lehame, S.A. Lerner, D.N., 2004a. Borehole Optimisation System (BOS) - a GIs based risk analysis tool for optimising the use of urban groundwater. Environmental Modelling and Software 19, 1111-1124]. This paper applies the BOS model to an urban Permo-Triassic Sandstone aquifer in the city centre of Nottingham, UK. The risk of pollution in potential new boreholes from the industrial chlorinated solvent tetrachloroethene (PCE) was assessed for this region. The risk model was validated against contaminant concentrations from 6 actual field boreholes within the study area. In these studies the model generally underestimated contaminant concentrations. A sensitivity analysis showed that the most responsive model parameters were recharge, effective porosity and contaminant degradation rate. Multiple simulations were undertaken across the study area in order to create surface maps indicating areas of low PCE concentrations, thus indicating the best locations to place new boreholes. Results indicate that northeastern, eastern and central regions have the lowest potential PCE concentrations in abstraction groundwater and therefore are the best sites for locating new boreholes. These locations coincide with aquifer areas that are confined by low permeability Mercia Mudstone deposits. Conversely southern and northwestern areas are unconfined and have shallower depth to groundwater. These areas have the highest potential PCE concentrations. These studies demonstrate the applicability of BOS as a tool for informing decision makers on the development of urban groundwater resources. (c) 2007 Elsevier Ltd. All rights reserved.