Insights into the biological significance of alternative splicing in Arabidopsis: functional characterization of a dual-targeted E3 ligase and the SCL30a SR protein

Dissertation presented to obtain the Ph.D degree in Molecular Biology

Alternative solvents in separation processes

Dissertation presented to obtain the Ph.D degree in Sustainable Chemistry

Femoral Approach: an Exceptional Alternative for Permanent Pacemaker Implantation

The classic transvenous implantation of a permanent pacemaker in a pectoral location may be precluded by obstruction of venous access through the superior vena cava or recent infection at the implant site. When these barriers to the procedure are bilateral and there are also contraindications or technical difficulties to performing a thoracotomy for an epicardial approach, the femoral vein, although rarely used, can be a viable alternative. We describe the case of a patient with occlusion of both subclavian veins and a high risk for mini-thoracotomy or videothoracoscopy, who underwent implantation of a permanent single-chamber pacemaker via the right femoral vein.

A Homemade Snare: An Alternative Method for Mechanical Removal of Dirofilaria Immitis in Dogs

Canine dirofilariosis is a life-threatening parasitic disease that is increasingly reported worldwide. Once diagnosed the main treatment goals are to improve the animal's clinical condition and to eliminate all life stages of the parasite with minimal posttreatment side effects. This can be achieved through mechanical, surgical, or chemotherapeutical approaches. Currently, manual extraction is the preferred method to remove adult heartworms due to its diminished invasiveness, reduced damage to the vascular endothelium, and shortened anaesthesia duration. However, it remains an expensive technique that can be highly traumatic. To address this issue, a nontraumatic homemade catheter-guided snare was developed for heartworm removal by adapting and folding a 0.014-inch coronary wire (BMW, Abbott Vascular). Transvenous heartworm extraction was performed on a dog severely infected with adult heartworms by inserting the modified snare into a 6-F Judkins right coronary guiding catheter BMW (Cordis) and advancing it into the right ventricle under fluoroscopic guidance. Fifteen adult specimens of Dirofilaria immitis were successfully extracted from the pulmonary artery and right ventricle without complications. To assure the death of both larvae and adults, postoperative treatment was successfully managed using ivermectin, doxycycline, and melarsomine, with no recurrence after surgery.

Validation of alternative additives for paste production

Dissertation presented to confer Master Degree in Chemical and Biochemical Engineering

An alternative antimonial schedule to be used in cutaneous leishmaniasis when high doses of antimony are undesirable

Despite more than half a century of use in leishmaniasis, antimony therapy still presents serious problems concerning dosage and toxicity. Low and high doses have been shown to be equally effective. In this paper, the feasibility of injecting one ampoule of meglumine antimoniate intramuscularly every other day until clinical cure is demonstrated, while studying a series of 40 cutaneous leishmaniasis cases. Total dose used varied from 1,822.5 to 12,150mg of pentavalent antimony and total time of treatment varied from 3 to 10 weeks, with 86% efficacy. Thirty-six out of the 40 patients are still on follow-up with a mean time of 10.7 ± 7 months and a median of 9 months. No relapse or mucosal lesions have been noted so far. The schedule showed good tolerance and easy application and its efficacy was comparable to the officially recommended WHO schedule. Therefore, such a schedule represents a valuable alternative for the cases with high toxicicity to antimony or daily injections are an obstacle to the treatment.

In vitro action of antiparasitic drugs, especially artesunate, against Toxoplasma gondii

INTRODUCTION: Toxoplasmosis is usually a benign infection, except in the event of ocular, central nervous system (CNS), or congenital disease and particularly when the patient is immunocompromised. Treatment consists of drugs that frequently cause adverse effects; thus, newer, more effective drugs are needed. In this study, the possible activity of artesunate, a drug successfully being used for the treatment of malaria, on Toxoplasma gondii growth in cell culture is evaluated and compared with the action of drugs that are already being used against this parasite. METHODS: LLC-MK2 cells were cultivated in RPMI medium, kept in disposable plastic bottles, and incubated at 36ºC with 5% CO2. Tachyzoites of the RH strain were used. The following drugs were tested: artesunate, cotrimoxazole, pentamidine, pyrimethamine, quinine, and trimethoprim. The effects of these drugs on tachyzoites and LLC-MK2 cells were analyzed using nonlinear regression analysis with Prism 3.0 software. RESULTS: Artesunate showed a mean tachyzoite inhibitory concentration (IC50) of 0.075µM and an LLC MK2 toxicity of 2.003µM. Pyrimethamine was effective at an IC50 of 0.482µM and a toxicity of 11.178µM. Trimethoprim alone was effective against the in vitro parasite. Cotrimoxazole also was effective against the parasite but at higher concentrations than those observed for artesunate and pyrimethamine. Pentamidine and quinine had no inhibitory effect over tachyzoites. CONCLUSIONS: Artesunate is proven in vitro to be a useful alternative for the treatment of toxoplasmosis, implying a subsequent in vivo effect and suggesting the mechanism of this drug against the parasite.

Characterization of an OPCPA laser amplifier based on the nonlinear crystal YCOB

The work described in this thesis was performed at the Laboratory for Intense Lasers (L2I) of Instituto Superior Técnico, University of Lisbon (IST-UL). Its main contribution consists in the feasibility study of the broadband dispersive stages for an optical parametric chirped pulse amplifier based on the nonlinear crystal yttrium calcium oxi-borate (YCOB). In particular, the main goal of this work consisted in the characterization and implementation of the several optical devices involved in pulse expansion and compression of the amplified pulses to durations of the order of a few optical cycles (20 fs). This type of laser systems find application in fields such as medicine, telecommunications and machining, which require high energy, ultrashort (sub-100 fs) pulses. The main challenges consisted in the preliminary study of the performance of the broadband amplifier, which is essential for successfully handling pulses with bandwidths exceeding 100 nm when amplified from the μJ to 20 mJ per pulse. In general, the control, manipulation and characterization of optical phenomena on the scale of a few tens of fs and powers that can reach the PW level are extremely difficult and challenging due to the complexity of the phenomena of radiation-matter interaction and their nonlinearities, observed at this time scale and power level. For this purpose the main dispersive components were characterized in detail, specifically addressing the demonstration of pulse expansion and compression. The tested bandwidths are narrower than the final ones, in order to confirm the parameters of these elements and predict the performance for the broadband pulses. The work performed led to additional tasks such as a detailed characterization of laser oscillator seeding the laser chain and the detection and cancelling of additional sources of dispersion.

The use of saliva as a practical and feasible alternative to urine in large-scale screening for congenital cytomegalovirus infection increasesinclusion and detection rates

INTRODUCTION: Although urine is considered the gold-standard material for the detection of congenital cytomegalovirus (CMV) infection, it can be difficult to obtain in newborns. The aim of this study was to compare the efficiency of detection of congenital CMV infection in saliva and urine samples. METHODS: One thousand newborns were included in the study. Congenital cytomegalovirus deoxyribonucleic acid (DNA) was detected by polymerase chain reaction (PCR). RESULTS: Saliva samples were obtained from all the newborns, whereas urine collection was successful in only 333 cases. There was no statistically significant difference between the use of saliva alone or saliva and urine collected simultaneously for the detection of CMV infection. CONCLUSIONS: Saliva samples can be used in large-scale neonatal screening for CMV infection.

Engineering bio-based polymers using alternative solvents and processes

The work presented in this thesis explores novel routes for the processing of bio-based polymers, developing a sustainable approach based on the use of alternative solvents such as supercritical carbon dioxide (scCO2), ionic liquids (ILs) and deep eutectic solvents (DES). The feasibility to produce polymeric foams via supercritical fluid (SCF) foaming, combined with these solvents was assessed, in order to replace conventional foaming techniques that use toxic and harmful solvents. A polymer processing methodology is presented, based on SCF foaming and using scCO2 as a foaming agent. The SCF foaming of different starch based polymeric blends was performed, namely starch/poly(lactic acid) (SPLA) and starch/poly(ε-caprolactone) (SPCL). The foaming process is based on the fact that CO2 molecules can dissolve in the polymer, changing their mechanical properties and after suitable depressurization, are able to create a foamed (porous) material. In these polymer blends, CO2 presents limited solubility and in order to enhance the foaming effect, two different imidazolium based ILs (IBILs) were combined with this process, by doping the blends with IL. The use of ILs proved useful and improved the foaming effect in these starch-based polymer blends. Infrared spectroscopy (FTIR-ATR) proved the existence of interactions between the polymer blend SPLA and ILs, which in turn diminish the forces that hold the polymeric structure. This is directly related with the ability of ILs to dissolve more CO2. This is also clear from the sorption experiments results, where the obtained apparent sorption coefficients in presence of IL are higher compared to the ones of the blend SPLA without IL. The doping of SPCL with ILs was also performed. The foaming of the blend was achieved and resulted in porous materials with conductivity values close to the ones of pure ILs. This can open doors to applications as self-supported conductive materials. A different type of solvents were also used in the previously presented processing method. If different applications of the bio-based polymers are envisaged, replacing ILs must be considered, especially due to the poor sustainability of some ILs and the fact that there is not a well-established toxicity profile. In this work natural DES – NADES – were the solvents of choice. They present some advantages relatively to ILs since they are easy to produce, cheaper, biodegradable and often biocompatible, mainly due to the fact that they are composed of primary metabolites such as sugars, carboxylic acids and amino-acids. NADES were prepared and their physicochemical properties were assessed, namely the thermal behavior, conductivity, density, viscosity and polarity. With this study, it became clear that these properties can vary with the composition of NADES, as well as with their initial water content. The use of NADES in the SCF foaming of SPCL, acting as foaming agent, was also performed and proved successful. The SPCL structure obtained after SCF foaming presented enhanced characteristics (such as porosity) when compared with the ones obtained using ILs as foaming enhancers. DES constituted by therapeutic compounds (THEDES) were also prepared. The combination of choline chloride-mandelic acid, and menthol-ibuprofen, resulted in THEDES with thermal behavior very distinct from the one of their components. The foaming of SPCL with THEDES was successful, and the impregnation of THEDES in SPCL matrices via SCF foaming was successful, and a controlled release system was obtained in the case of menthol-ibuprofen THEDES.

The potential field method and the nonlinear attractor dynamics approach: what are the differences?

One of the most popular approaches to path planning and control is the potential field method. This method is particularly attractive because it is suitable for on-line feedback control. In this approach the gradient of a potential field is used to generate the robot's trajectory. Thus, the path is generated by the transient solutions of a dynamical system. On the other hand, in the nonlinear attractor dynamic approach the path is generated by a sequence of attractor solutions. This way the transient solutions of the potential field method are replaced by a sequence of attractor solutions (i.e., asymptotically stable states) of a dynamical system. We discuss at a theoretical level some of the main differences of these two approaches.

Seismic vulnerability of existing masonry buildings: Nonlinear parametric analysis

Existing masonry structures are usually associated to a high seismic vulnerability, mainly due to the properties of the materials, weak connections between floors and load-bearing walls, high mass of the masonry walls and flexibility of the floors. For these reasons, the seismic performance of existing masonry structures has received much attention in the last decades. This study presents the parametric analysis taking into account the deviations on features of the gaioleiro buildings - Portuguese building typology. The main objective of the parametric analysis is to compare the seismic performance of the structure as a function of the variations of its properties with respect to the response of a reference model. The parametric analysis was carried out for two types of structural analysis, namely for the non-linear dynamic analysis with time integration and for the pushover analysis with distribution of forces proportional to the inertial forces of the structure. The Young's modulus of the masonry walls, Young's modulus of the timber floors, the compressive and tensile non-linear properties (strength and fracture energy) were the properties considered in both type of analysis. Additionally, in the dynamic analysis, the influences of the vis-cous damping and of the vertical component of the earthquake were evaluated. A pushover analysis proportional to the modal displacement of the first mode in each direction was also carried out. The results shows that the Young's modulus of the masonry walls, the Young's modulus of the timber floors and the compressive non-linear properties are the pa-rameters that most influence the seismic performance of this type of tall and weak existing masonry structures. Furthermore, it is concluded that that the stiffness of the floors influences significantly the strength capacity and the collapse mecha-nism of the numerical model. Thus, a study on the strengthening of the floors was also carried out. The increase of the thickness of the timber floors was the strengthening technique that presented the best seismic performance, in which the reduction of the out-of-plane displacements of the masonry walls is highlighted.

Recycled asphalt mixtures with foamed bitumen: an alternative to build ecofriendly road pavements

Nowadays, recycling has become a very important objective for the society in the scope of a closed loop product life cycle. In recent years, new recycling techniques have been developed in the area of road pavements that allow the incorporation of high percentages of reclaimed asphalt (RA) materials in recycled asphalt mixtures. The use of foamed bitumen for production of recycled asphalt mixtures is one of those techniques, which also allows the reduction of the mixing temperatures (warm mix technology). However, it is important to evaluate if this solution can maintain or improve the performance of the resulting mixtures. Thus, the main aim of the present study is to assess the performance of warm recycled asphalt mixtures incorporating foamed bitumen as the new binder and 50% RA, in comparison with a control mixture using conventional bitumen. Four mixtures have been produced with 50% RA, one of them at typical high mixing temperatures with a conventional bitumen (control mixture) and the other three with foamed bitumen at different production temperatures. These four mixtures were tested to evaluate their compactability and water sensitivity. The laboratory test results showed that the production of recycled mixtures with foamed bitumen can be reduced by 40ºC without changing the performance of the resulting mixtures.

Foamed Bitumen: an alternative way of producing asphalt mixtures

With the constant need to improve and make the production of asphalt mixtures more sustainable, new production techniques have been developed, the implementation of which implies the correct knowledge of their performance. One of the most promising asphalt production techniques is the use of foamed bitumen. However, it is essential to understand how this binder will behave when subject to the expansion process. The loss of volume of the foamed bitumen could be translated by a decay curve, which allows to determine the ideal temperature and water content added to the bitumen in order to assure adequate conditions to the mix the bitumen with the aggregates. On the present study, a conventional 160/220 pen grade bitumen was tested by using different temperatures and water contents, and it was concluded that the optimum temperature for the production of foamed bitumen (with the studied bitumen) is 150 ºC, which corresponds to a viscosity of 0.1 Pa.s. The water content mostly influence the half-life of the bitumen foam, resulting in quicker volume reductions for higher water contents.

Evaluation of seismic vulnerability assessment parameters for portuguese vernacular constructions with nonlinear numerical analysis

Considering that vernacular architecture may bear important lessons on hazard mitigation and that well-constructed examples showing traditional seismic resistant features can present far less vulnerability than expected, this study aims at understanding the resisting mechanisms and seismic behavior of vernacular buildings through detailed finite element modeling and nonlinear static (pushover) analysis. This paper focuses specifically on a type of vernacular rammed earth constructions found in the Portuguese region of Alentejo. Several rammed earth constructions found in the region were selected and studied in terms of dimensions, architectural layout, structural solutions, construction materials and detailing and, as a result, a reference model was built, which intends to be a simplified representative example of these constructions, gathering the most common characteristics. Different parameters that may affect the seismic response of this type of vernacular constructions have been identified and a numerical parametric study was defined aiming at evaluating and quantifying their influence in the seismic behavior of this type of vernacular buildings. This paper is part of an ongoing research which includes the development of a simplified methodology for assessing the seismic vulnerability of vernacular buildings, based on vulnerability index evaluation methods.