Application of ionic liquids and enzymes for the removal of proteinaceous layers from polychrome of works of art and evaluation of the cleaning effectiveness

Dissertação Apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Ciências da Conservação, especialização em Pintura

Health-Related Quality of Life Impact of a Triple Combination of Olmesartan Medoxomil, Amlodipine Besylate and Hydrochlorotiazide in Subjects with Hypertension

BACKGROUND: A post-hoc analysis was performed on the data from a 54 weeks phase III study (ClinicalTrials.gov identifier: NCT00923091) to measure changes in the health-related quality of life (HRQoL) of 2,690 patients aged ≥18 with moderate-to-severe hypertension who received one of six doses of olmesartan/amlodipine/hydrochlorothiazide (OLM/AML/HCTZ), using the MINICHAL and EQ-5D instruments. METHODS: Descriptive statistics were used to assess blood pressure and HRQoL scores over the study period. Analysis of covariance (ANCOVA) was used to identify those factors that could possibly have influenced HRQoL. Linear regression was used to assess the relationship between changes in blood pressure and HRQoL scores. RESULTS: Patients' baseline MINICHAL mood and somatic domains scores were 5.5 and 2.6. Over the study period HRQoL improved as both MINICHAL scores decreased by 31-33%. Patients' baseline EQ-5D index and VAS scores were 0.9 and 73.4 respectively, increasing by 6% and 12% over the study period. Patients' QALY gain over the 54 weeks study period was estimated to be 0.029 QALYs. The ANCOVA showed that changes in patients' HRQoL was likely to have been influenced by patients' achievement of blood pressure control, the amount of concomitant medication and patients' last used dosage strength of antihypertensive. Linear regression showed that blood pressure improvement may have been associated with improved HRQoL. CONCLUSIONS: This study showed that OLM/AML/HCTZ reduced blood pressure and significantly increased blood pressure control whilst improving patients' HRQoL. Achieving blood pressure control, amount of concomitant medication and dosage strength of antihypertensive impacted on patients' HRQoL.

New roles of Rab GTPases on eye diseases: targeting VEGF secretion

RESUMO: A retina é composta, entre outras estruturas, pelo epitélio pigmentar da retina (EPR)e pela coróide. A região central da retina denomina-se mácula, e é a zona mais afetada na degenerescência macular relacionada com a idade, a forma mais comum de degenerescência da retina. Nesta doença, a secreção de fatores de crescimento pelo EPR é afetada, nomeadamente a do fator de crescimento vascular endotelial (VEGF), e pouco se sabe ainda sobre os mecanismos moleculares conducentes a esta condição. A família de proteínas Rab GTPases está envolvida nas vias intracelulares de sinalização e tráfego membranares, essenciais na transdução de sinais extracelulares em respostas biológicas. A sua crucial importância nestes mecanismos levou-nos a considerar o seu potencial envolvimento nas vias de secreção do VEGF, e a questionar-nos se teriam algum papel regulador sobre as mesmas. O principal objetivo deste trabalho é identificar Rab GTPases importantes para as vias de secreção e endocitose do VEGF no EPR. Essa identificação ajudará a esclarecer a patogénese da degenerescência macular da retina, e poderá servir para uma procura mais direcionada de novos agentes terapêuticos. A caracterização de dois modelos in vitro do EPR, células primárias isoladas de murganho e a linha celular B6-RPE07,levou-nos a concluir que são ambos semelhantes. Contudo, a linha celular foi escolhida como protótipo do EPR por permitir o acesso a um número ilimitado de células. No decurso deste trabalho, desenvolvemos e caracterizámos uma biblioteca de ferramentas moleculares que nos permitiram reduzir os níveis proteicos das proteínas Rab GTPases, com base na tecnologia de ácido ribonucleico (ARN) de interferência. O papel das proteínas Rab GTPases na secreção do VEGF no EPR foi estudado com base no silenciamento de apenas uma proteína, ou combinando várias, segundo a sua localização e funções intracelulares descritas. Este trabalho permitiu-nos concluir que as proteínas Rab GTPases são importantes intervenientes no processo de secreção de VEGF pelo EPR, e confirmar dados anteriores que relatam o envolvimento de algumas Rab GTPases endocíticas no processo. Propomos ainda um novo modelo para a interação destas proteínas no EPR, e sugerimos que a Rab10 e a Rab14 atuam negativamente sobre a Rab8, controlando o seu funcionamento. Os nossos resultados evidenciam a importância das proteínas Rab GTPases na secreção do VEGF pelas células do EPR, e servem de base a futuros estudos que melhor procurem compreender este mecanismo e de que modo a sua alteração se relaciona com a degenerescência da retina.--------ABSTRACT: Retinal pigment epithelium (RPE) and choroid are components of the mammalian retina, of which the central region is called macula. The most common form of retinaldegeneration, age-related macular degeneration (AMD), involves primarily deregulation of growth factors secretion by the RPE. Very little is known about the molecular mechanisms that lead to impairment of RPE’s homeostatic intracellular processes, namely the secretion of vascular endothelial growth factor (VEGF). Rab GTPases’ family regulates membrane targeting and traffic, being essential in the transduction of signal pathways. Given Rab proteins’ role in intracellular trafficking, we propose to identify key regulatory Rab proteins involved in either the secretory or the recycling pathways of VEGF in RPE. Understanding how Rab proteins’ function disruption could lead to retinal and choroidal pathology would ultimately contribute to find new therapeutic agents. Here, we characterized two mouse RPE in vitro cell models, primary cells and B6-RPE07 cell line, and concluded that both display important epithelial features as the RPE presents in vivo. Considering unlimited cell number and results reproducibility, we chose B6-RPE07 cells to further study Rab proteins’ function. To scrutinize the consequences of Rab proteins’ absence or diminished levels, we have developed novel molecular tools to achieve silencing of these key proteins using miRNA technology. We further addressed the effect of Rab proteins’ absence on VEGF secretion by performing an extensive screening where different Rab proteins were silenced, both individually and in multiple combinations considering their cellular/ compartment location. We conclude that Rab GTPases are important intervenients in VEGF secretion by RPE cells, confirming endocytic Rab proteins’ role in regulation of VEGF biology. We also propose a novel model for Rab proteins’ interaction in RPE. Our results suggest that Rab10 and Rab14 might influence Rab8 in a negative feedback mechanism, important for controlling VEGF secretion. Our achievements’ unravel Rab proteins’ role in VEGF secretion by RPE cells and are the basis for future studies to better understand RPE molecular secretory machinery.

Cape Verde’s foreign policy: an economic perspective

The virtuous cycle between development success and foreign policy in Cape Verde reflects a positive interaction between globalization and governance. Development success under globalization entails positive market perceptions regarding the orientation and predictability of policies as well as the accompanying institutional arrangements, thereby making foreign policy salient beyond the comparator group, or “aspirational”. Even if there is no universally applicable development model, an aspirational foreign policy can be built on positive rankings with respect to comparator groups. In Macedo and Pereira (2010), macrolevel policy and institutional combinations underpinning trade diversification and income convergence in West and Southern Africa are used to establish development success for Cape Verde and Mozambique respectively. Here, the narrative of long-term development helps identify the following drivers: moving towards a market economy; opening up to regional and global trade; increasing economic and political freedom; pursuing macroeconomic stability and financial reputation; ensuring policy continuity (especially in trade and industrial sectors) and focusing on human development (especially poverty reduction and education). Looking at GDP per capita and indicators of financial reputation and good governance of sub-regional peers is not sufficient to conclude that Cape Verde’s convergence will be sustained. Nevertheless, the positive interaction between trade and financial globalization, on the one hand, and democracy and good governance, on the other, have positive implications for the effectiveness of foreign policy across the region as well as in the Portuguese-speaking community.

Porous structures for the purification of biopharmaceuticals

This work aimed at the development of a (bio)polymeric monolithic support for biopharmaceuticals purification and/or capture. For that, it was assured that functional groups on its surface were ready to be involved in a plethora of chemical reactions for incorporation of the desired and most suitable ligand. Using cryogelation as preparation method a screening on multiple combinations of materials was performed in order to create a potentially efficient support with the minimal footprint, i.e. a monolithic support with reasonable mechanical properties, highly permeable, biocompatible, ready to use, with gravitational performance and minimal unspecific interactions towards the target molecules, but also biodegradable and produced from renewable materials. For the pre-selection all monoliths were characterized physico-chemically and morphologically; one agarose-based and two chitosan-based monoliths were then subjected to further characterizations before and after their modification with magnetic nanoparticles. These three specimens were finally tested towards adenovirus and the recovery reached 84% for the chitosan-GMA plain monolith prepared at -80°C. Monoliths based on chitosan and PVA were prepared in the presence and absence of magnetic particles, and tested for the isolation of GFP directly from crude cellular extracts. The affinity ligand A4C7 previously selected for GFP purification was synthesized on the monolith. The results indicated that the solid-phase synthesis of the ligand directly onto the monolith might require optimization and that the large pores of the monoliths are unsuitable for the purification of small proteins, such as GFP.

Wireless triple play system

Dissertação para obtenção do Grau de Mestre em Engenharia Electrotécnica e Computadores

G and P rotavirus genotypes in stool samples from children in Teresina, State of Piauí

A total of 123 stool specimens collected in Teresina, Piauí between 1994 and 1996, from 0 to 2-year-old children with diarrhea, were used for this study. Molecular characterization of the G and P rotavirus genotypes was performed using the reverse transcriptase polymerase chain reaction. The following results were obtained for the P genotypes: P[8] (17. 1%), P[1] (4. 9%), P[4] (3. 3%), P[6, M37] (2. 4%) and mixtures (27. 6%). The P[1]+P[8] mixture was found in 19. 5% of the samples. For the G genotypes, the results were: G1 (25. 2%), G5 (13. 8%), G2 (2. 5%), G4 (2. 5%), G9 (0. 8%) and mixtures (41. 5%). G1+G5 was the mixture most frequently found (12. 1%). Our results showed unusual combinations such as P[1]G5 and P[1]+P[8]G5. The high percentage of mixtures and unusual combinations containing mixtures of human and animal rotavirus genotypes strongly suggests the possibility of gene reassortment and interspecies transmission.

International meeting: new diagnostic tests are urgently needed to treat patients with Chagas disease

Trypanosoma cruzi infection is often not detected early on or actively diagnosed, partly because most infected individuals are either asymptomatic or oligosymptomatic. Moreover, in most places, neither blood banks nor healthcare units offer diagnostic confirmation or treatment access. By the time patients present clinical manifestations of advanced chronic Chagas disease, specific treatment with current drugs usually has limited effectiveness. Better-quality serological assays are urgently needed, especially rapid diagnostic tests for diagnosis patients in both acute and chronic phases, as well as for confirming that a parasitological cure has been achieved. Some new antigen combinations look promising and it is important to assess which ones are potentially the best, together with their requirements in terms of investigation and development. In August 2007, a group of specialized researchers and healthcare professionals met to discuss the state of Chagas infection diagnosis and to build a consensus for a plan of action to develop efficient, affordable, accessible and easy-to-use diagnostic tests for Chagas disease. This technical report presents the conclusions from that meeting.

Tool for discovering sequential patterns in financial markets

The goal of this thesis is the study of a tool that can help analysts in finding sequential patterns. This tool will have a focus on financial markets. A study will be made on how new and relevant knowledge can be mined from real life information, potentially giving investors, market analysts, and economists new basis to make informed decisions. The Ramex Forum algorithm will be used as a basis for the tool, due to its ability to find sequential patterns in financial data. So that it further adapts to the needs of the thesis, a study of relevant improvements to the algorithm will be made. Another important aspect of this algorithm is the way that it displays the patterns found, even with good results it is difficult to find relevant patterns among all the studied samples without a proper result visualization component. As such, different combinations of parameterizations and ways to visualize data will be evaluated and their influence in the analysis of those patterns will be discussed. In order to properly evaluate the utility of this tool, case studies will be performed as a final test. Real information will be used to produce results and those will be evaluated in regards to their accuracy, interest, and relevance.

Dissecting gastrointestinal dysfunction and inflammation in a “pre-motor” rodent model of Parkinson’s disease

Research on Parkinson’s disease (PD) has mainly focused on the degeneration of the dopaminergic neurons of nigro-striatal (NS) pathway; also, post-mortem studies have demonstrated that the noradrenergic and the serotonergic transmitter systems are also affected (Jellinger, 1999). Degeneration of these neuronal cell bodies is generally thought to start prior to the loss of dopaminergic neurons in the NS pathway and precedes the appearance of the motor symptoms that are the “hallmark” of PD. Gastrointestinal (GI) motility is often disturbed in PD, manifesting chiefly as impaired gastric emptying and constipation. These GI dysfunction symptoms may be the result of a loss in noradrenergic and serotonergic innervation. GI deficits were evaluated using an organ bath technique. Groups treated with different combinations of neurotoxins (6-OHDA alone, 6-OHDA + pCA or 6-OHDA + DSP-4) presented significant differences in gut contractility compared to control groups. Since a substantial body of literature suggests the presence of an inflammatory process in parkinsonian state (Whitton, 2007), changes in pro-inflammatory cytokines in the gut were assessed using a cytokine microarray. It has been found in this work that groups with a combined dopaminergic and noradrenergic lesion have a significant increase in both expressions of IL-13 and VEGF. IL-6 also shows a decrease in treatment groups; however this decrease did not reach statistical significance. The therapeutic value of Exendin-4 (EX-4) was evaluated. It has been previously demonstrated that EX-4, a glucagon-like peptide-1 receptor (GLP-1R) agonist, is neuroprotective in rodent models of PD (Harkavyi et al., 2008). In this thesis it has been found that EX-4 was able to reverse a decrease in gut contractility obtained through intracerebral bilateral 6-OHDA injection. Although more studies are required, EX-4 could be used as a possible therapy for the GI symptoms prominent in the early stages of PD.

Study of the electromyographic signal dynamic behavior in Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by motor neurons degeneration, which reduces muscular force, being very difficult to diagnose. Mathematical methods are used in order to analyze the surface electromiographic signal’s dynamic behavior (Fractal Dimension (FD) and Multiscale Entropy (MSE)), evaluate different muscle group’s synchronization (Coherence and Phase Locking Factor (PLF)) and to evaluate the signal’s complexity (Lempel-Ziv (LZ) techniques and Detrended Fluctuation Analysis (DFA)). Surface electromiographic signal acquisitions were performed in upper limb muscles, being the analysis executed for instants of contraction for ipsilateral acquisitions for patients and control groups. Results from LZ, DFA and MSE analysis present capability to distinguish between the patient group and the control group, whereas coherence, PLF and FD algorithms present results very similar for both groups. LZ, DFA and MSE algorithms appear then to be a good measure of corticospinal pathways integrity. A classification algorithm was applied to the results in combination with extracted features from the surface electromiographic signal, with an accuracy percentage higher than 70% for 118 combinations for at least one classifier. The classification results demonstrate capability to distinguish members between patients and control groups. These results can demonstrate a major importance in the disease diagnose, once surface electromyography (sEMG) may be used as an auxiliary diagnose method.

Digital microfluidic devices: the role of the dielectric layer

Digital microfluidics (DMF) is a field which has emerged in the last decade as a re-liable and versatile tool for sensing applications based on liquid reactions. DMF allows the discrete displacement of droplets, over an array of electrodes, by the application of voltage, and also the dispensing from a reservoir, mixing, merging and splitting fluidic operations. The main drawback of these devices is due to the need of high driving volt-ages for droplet operations. In this work, alternative dielectric layers combinations were studied aiming the reduction of these driving voltages. DMF chips were designed, pro-duced and optimized according to the theory of electrowetting-on-dielectric, adopting different combinations of parylene-C and tantalum pentoxide (Ta2O5) as dielectric ma-terials, and Teflon as hydrophobic layer. With both devices’ configurations, i.e., Parylene as single dielectric, and multilayer chips combining Parylene and Ta2O5, it was possible to perform all the fluidic opera-tions in the microliter down to hundreds of nanoliters range. Multilayer chips presented significant reduction on driving voltages for droplet op-erations in silicone oil filler medium: from 70 V (parylene only) down to 30 V (parylene/Ta2O5) for dispensing; and from 50 V (parylene only) down to 15 V (parylene/Ta2O5) for movement. Peroxidase colorimetric reactions were successfully performed as proof-of-concept, using multilayer configuration devices.

Structured products in the current low interest rate environment

Directed Research Internship

Assessment of microbiological air quality in hemato-oncology units and its relationship with the occurrence of invasive fungal infections: an integrative review

Worldwide aging of the human population has promoted an increase in the incidence of neoplasia, including hematological cancers, which render patients particularly vulnerable to invasive fungal infections. For this reason, air filtration in hemato-oncology units has been recommended. However, scarce literature has assessed the impact of microbiological air quality on the occurrence of fungal infections in this population. We performed an integrative review of studies in the MEDLINE database that were published between January 1980 and October 2012, using the following combinations of keywords: air × quality × HEPA, air × quality × hematology, and airborne fungal infections. The search yielded only 13 articles, suggesting that high-efficiency filtering of the ambient air in hemato-oncology units can prevent the incidence of invasive fungal infections. However, no randomized clinical trial was found to confirm this suggestion. Currently, there is no consensus about the maximum allowable count of fungi in the air, which complicates filtration monitoring, including filter maintenance and replacement, and needs to be addressed in future studies.

Blood feeding patterns of Nyssomyia intermedia and Nyssomyia neivai (Diptera, Psychodidae) in a cutaneous leishmaniasis endemic area of the Ribeira Valley, State of São Paulo, Brazil

Introduction The aim of this study was to identify the blood feeding sources of Nyssomyia intermedia (Ny. intermedia) and Nyssomyia neivai (Ny. neivai), which are Leishmania vectors and the predominant sandfly species in the Ribeira Valley, State of São Paulo, Brazil, an endemic area for cutaneous leishmaniasis. Methods Specimens were captured monthly between February 2001 and December 2003 on a smallholding and a small farm situated in the Serra district in the Iporanga municipality. The blood meals of 988 engorged females were tested using the avidin-biotin immunoenzymatic enzyme-linked immunosorbent assay (ELISA). Seven blood meal sources were investigated: human, dog, chicken, bovine, pig, horse and rat. Results The results showed that among the females that fed on one or more blood sources, the respective percentages for Ny. intermedia and Ny. neivai, respectively, were as follows: human (23% and 36.8%), pig (47.4% and 26.4%), chicken (25.7% and 36.8%) and dog (3.9% and 0%), and the differences in the blood sources between the two species were statistically significant (p = 0.043). Conclusions Both species had predominant reactivity for one or two blood sources, and few showed reactivity indicating three or four sources. Many different combinations were observed among the females that showed reactivity for more than one source, which indicated their opportunistic habits and eclecticism regarding anthropic environmental conditions.