Cohesion decay: quantitative analysis of partial sister chromatid cohesion

Cell division is a highly dynamic process where sister chromatids remain associated with each other from the moment of DNA replication until the later stages of mitosis, giving rise to two daughter cells with equal genomes. The “molecular glue” that links sister DNA molecules is called cohesin, a tripartite ring-like protein complex composed of two Structural Maintenance of Chromosome proteins (Smc1 and Smc3) bridged by a kleisin subunit Rad21/Scc1, that together prevent precocious sister chromatid separation. Accumulating evidence has suggested that cohesion decay may be the cause of segregation errors that underlie certain human pathologies. However it remains to be determined how much cohesin loss abolishes functional sister chromatid cohesion. To answer these questions, we have developed different experimental conditions aiming to titrate the levels of cohesin on mitotic chromosomes in a precise manner. Using these tools, we will determine the minimal amount of cohesin needed to confer functional cohesion. The approaches described here take advantage of a system in Drosophila melanogaster where the Tobacco Etch Virus (TEV) protease can cleave the Rad21 subunit of cohesin leading to precocious sister chromatid separation. Firstly, we tried to express different levels of TEV protease to obtain partial loss of cohesion. However, this approach has failed to produce systematic different levels of sister chromatid separation. Most of the work was therefore focused on a second strategy, for which we established strains with different levels of cohesin sensitive/cohesin resistant to TEV protease. Strains containing different amounts of functional cohesin (TEV resistant) were tested by in vitro cleavage and by in vivo injections in embryos for their ability to promote sister chromatid cohesion. Our results reveal that removal of half of the cohesin complexes does not impair chromosome segregation, implying that chromosome cohesion is less sensitive to cohesin amounts than previously anticipated.

Application of NMR RDC’s, relaxation and self-diffusion for the study of dynamic processes of small molecules in solution

Spin-lattice Relaxation, self-Diffusion coefficients and Residual Dipolar Couplings (RDC’s) are the basis of well established Nuclear Magnetic Resonance techniques for the physicochemical study of small molecules (typically organic compounds and natural products with MW < 1000 Da), as they proved to be a powerful and complementary source of information about structural dynamic processes in solution. The work developed in this thesis consists in the application of the earlier-mentioned NMR techniques to explore, analyze and systematize patterns of the molecular dynamic behavior of selected small molecules in particular experimental conditions. Two systems were chosen to investigate molecular dynamic behavior by these techniques: the dynamics of ion-pair formation and ion interaction in ionic liquids (IL) and the dynamics of molecular reorientation when molecules are placed in oriented phases (alignment media). The application of NMR spin-lattice relaxation and self-diffusion measurements was applied to study the rotational and translational molecular dynamics of the IL: 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4]. The study of the cation-anion dynamics in neat and IL-water mixtures was systematically investigated by a combination of multinuclear NMR relaxation techniques with diffusion data (using by H1, C13 and F19 NMR spectroscopy). Spin-lattice relaxation time (T1), self-diffusion coefficients and nuclear Overhauser effect experiments were combined to determine the conditions that favor the formation of long lived [BMIM][BF4] ion-pairs in water. For this purpose and using the self-diffusion coefficients of cation and anion as a probe, different IL-water compositions were screened (from neat IL to infinite dilution) to find the conditions where both cation and anion present equal diffusion coefficients (8% water fraction at 25 ºC). This condition as well as the neat IL and the infinite dilution were then further studied by 13C NMR relaxation in order to determine correlation times (c) for the molecular reorientational motion using a mathematical iterative procedure and experimental data obtained in a temperature range between 273 and 353 K. The behavior of self-diffusion and relaxation data obtained in our experiments point at the combining parameters of molar fraction 8 % and temperature 298 K as the most favorable condition for the formation of long lived ion-pairs. When molecules are subjected to soft anisotropic motion by being placed in some special media, Residual Dipolar Couplings (RDCs), can be measured, because of the partial alignment induced by this media. RDCs are emerging as a powerful routine tool employed in conformational analysis, as it complements and even outperforms the approaches based on the classical NMR NOE or J3 couplings. In this work, three different alignment media have been characterized and evaluated in terms of integrity using 2H and 1H 1D-NMR spectroscopy, namely the stretched and compressed gel PMMA, and the lyotropic liquid crystals CpCl/n-hexanol/brine and cromolyn/water. The influence that different media and degrees of alignment have on the dynamic properties of several molecules was explored. Different sized sugars were used and their self-diffusion was determined as well as conformation features using RDCs. The results obtained indicate that no influence is felt by the small molecules diffusion and conformational features studied within the alignment degree range studied, which was the 3, 5 and 6 % CpCl/n-hexanol/brine for diffusion, and 5 and 7.5 % CpCl/n-hexanol/brine for conformation. It was also possible to determine that the small molecules diffusion verified in the alignment media presented close values to the ones observed in water, reinforcing the idea of no conditioning of molecular properties in such media.

Dynamic stiffness of the piled foundation of a onshore wind turbine

One of today's biggest concerns is the increase of energetic needs, especially in the developed countries. Among various clean energies, wind energy is one of the technologies that assume greater importance on the sustainable development of humanity. Despite wind turbines had been developed and studied over the years, there are phenomena that haven't been yet fully understood. This work studies the soil-structure interaction that occurs on a wind turbine's foundation composed by a group of piles that is under dynamic loads caused by wind. This problem assumes special importance when the foundation is implemented on locations where safety criteria are very demanding, like the case of a foundation mounted on a dike. To the phenomenon of interaction between two piles and the soil between them it's given the name of pile-soil-pile interaction. It is known that such behavior is frequency dependent, and therefore, on this work evaluation of relevant frequencies for the intended analysis is held. During the development of this thesis, two methods were selected in order to assess pile-soil-pile interaction, being one of analytical nature and the other of numerical origin. The analytical solution was recently developed and its called Generalized pile-soil-pile theory, while for the numerical method the commercial nite element software PLAXIS 3D was used. A study of applicability of the numerical method is also done comparing the given solution by the nite element methods with a rigorous solution widely accepted by the majority of the authors.

Analysis and treatment of a nineteenth century oil painting

This thesis focused on the study and treatment of a 19th century female portrait in oil from ECOMUSEU Municipal do Seixal, Portugal. The portrait, which depicts Isabel Maria Lourenço Affonso was in poor condition and a large strip of paint and canvas was missing (approximately 9cm by 66cm, almost 11% of the total surface area). The portrait is a companion piece to a male portrait (the relationship was established as part of this study), therefore a technical study of both paintings was considered essential to support the choices made during the treatment. The project involved three main areas: - The study of the history, condition, materials and techniques of both paintings. This allowed their comparison and understanding of their relationship; - The treatment of Isabel Maria Lourenço Affonso. The choices made and problems encountered are described. - The production of a replacement for the missing strip of paint and canvas. The practical solution developed to overcome such an unusual challenge is described along with the creative and flexible thinking required. Because not all traditional infill materials cope well on a mechanical level with thin layers over a very large surface (many are too brittle), strict criteria had to be employed to choose the appropriate material. The primary goal was to find a fill which would remain flexible and be capable of accepting surface texture, such that there would be a good visual match with the painting. Analysis and testing was carried out to evaluate the physical properties of the fill material chosen, BEVA® Gesso-P. The successful creation of the replacement strip has resulted in two publications and one presentation: Publication pending in The Picture Restorer, Leslie Carlyle, Raquel Marques, Isabel Pombo Cardoso and Sara Babo, “Creating a Textured Replacement Strip for the Missing Lower Portion of an Oil Portrait: Problem Solving and Practical Solutions”. Abstract accepted for presentation and publication, International Meeting on Retouching of Cultural Heritage (2RECH), Raquel Marques, Leslie Carlyle and Isabel Pombo Cardoso, “Textured Replacement Strip for a Missing Portion of a Portrait: Problem Solving and Practical Solutions”.

Mechanical characterization of aortas using 2D ultrasound elastography

Rupture of aortic aneurysms (AA) is a major cause of death in the Western world. Currently, clinical decision upon surgical intervention is based on the diameter of the aneurysm. However, this method is not fully adequate. Noninvasive assessment of the elastic properties of the arterial wall can be a better predictor for AA growth and rupture risk. The purpose of this study is to estimate mechanical properties of the aortic wall using in vitro inflation testing and 2D ultrasound (US) elastography, and investigate the performance of the proposed methodology for physiological conditions. Two different inflation experiments were performed on twelve porcine aortas: 1) a static experiment for a large pressure range (0 – 140 mmHg); 2) a dynamic experiment closely mimicking the in vivo hemodynamics at physiological pressures (70 – 130 mmHg). 2D raw radiofrequency (RF) US datasets were acquired for one longitudinal and two cross-sectional imaging planes, for both experiments. The RF-data were manually segmented and a 2D vessel wall displacement tracking algorithm was applied to obtain the aortic diameter–time behavior. The shear modulus G was estimated assuming a Neo-Hookean material model. In addition, an incremental study based on the static data was performed to: 1) investigate the changes in G for increasing mean arterial pressure (MAP), for a certain pressure difference (30, 40, 50 and 60 mmHg); 2) compare the results with those from the dynamic experiment, for the same pressure range. The resulting shear modulus G was 94 ± 16 kPa for the static experiment, which is in agreement with literature. A linear dependency on MAP was found for G, yet the effect of the pressure difference was negligible. The dynamic data revealed a G of 250 ± 20 kPa. For the same pressure range, the incremental shear modulus (Ginc) was 240 ± 39 kPa, which is in agreement with the former. In general, for all experiments, no significant differences in the values of G were found between different image planes. This study shows that 2D US elastography of aortas during inflation testing is feasible under controlled and physiological circumstances. In future studies, the in vivo, dynamic experiment should be repeated for a range of MAPs and pathological vessels should be examined. Furthermore, the use of more complex material models needs to be considered to describe the non-linear behavior of the vascular tissue.

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.

Analysis and visualization of energy use for university campus

The reduction of greenhouse gas emissions is one of the big global challenges for the next decades due to its severe impact on the atmosphere that leads to a change in the climate and other environmental factors. One of the main sources of greenhouse gas is energy consumption, therefore a number of initiatives and calls for awareness and sustainability in energy use are issued among different types of institutional and organizations. The European Council adopted in 2007 energy and climate change objectives for 20% improvement until 2020. All European countries are required to use energy with more efficiency. Several steps could be conducted for energy reduction: understanding the buildings behavior through time, revealing the factors that influence the consumption, applying the right measurement for reduction and sustainability, visualizing the hidden connection between our daily habits impacts on the natural world and promoting to more sustainable life. Researchers have suggested that feedback visualization can effectively encourage conservation with energy reduction rate of 18%. Furthermore, researchers have contributed to the identification process of a set of factors which are very likely to influence consumption. Such as occupancy level, occupants behavior, environmental conditions, building thermal envelope, climate zones, etc. Nowadays, the amount of energy consumption at the university campuses are huge and it needs great effort to meet the reduction requested by European Council as well as the cost reduction. Thus, the present study was performed on the university buildings as a use case to: a. Investigate the most dynamic influence factors on energy consumption in campus; b. Implement prediction model for electricity consumption using different techniques, such as the traditional regression way and the alternative machine learning techniques; and c. Assist energy management by providing a real time energy feedback and visualization in campus for more awareness and better decision making. This methodology is implemented to the use case of University Jaume I (UJI), located in Castellon, Spain.

Assessment by doppler ultrasound of entheseal lesions in spondyloarthritis : a longitudial study to determine structural damage and disease activity lesions

RESUMO: Enthesitis is the hallmark of spondyloarthritis (SpA), and is observed in all subtypes. Wide information on SpA abnormalities, including synovitis, tendinitis and enthesitis, can be efficiently perceived by Doppler ultrasound. Furthermore, several studies on imaging of enthesis showed that imaging techniques are better than clinical examination to detect enthesis alterations; and vascularized enthesitis detected by Doppler ultrasound appears to be a valuable diagnostic tool to confirm SpA diagnosis. However, data published until now concerning entheseal elementary alterations that characterize SpA enthesitis (enthesis inflammatory activity) or enthesopathy (permanent structural changes) reflect rather the authors’ empiric opinion than a methodological validation process. In this sense it seems crucial to identify elementary entheseal lesions associated with activity or damage, in order to improve monitoring and treatment response in SpA patients. The development of better assessment tools is today a challenge and a need in SpA. The first study of this thesis focused on the analysis of the reliability of inter-lector and inter-ultrasonography equipment of Madrid sonography enthesitis index (MASEI). Fundamental data for the remaining unrolling project validity. In the second and third studies we concerned about two entheseal elemental lesions: erosions and bursa. In literature erosions represent a permanent structural damage, being useful for monitoring joint injury, disease activity and therapeutic response in many rheumatic diseases; and to date, this concept has been mostly applied in rheumatoid arthritis (RA). Unquestionably, erosion is a tissue-related damage and a structural change. However, the hypothesis that we decided to test was if erosions represent a permanent structural change that can only grow and worsen over time, as occurs in RA, or a transitory alteration. A longitudinal study of early SpA patients was undertaken, and the Achilles enthesis was used as a model. Our results strongly suggested that previously detected erosions could disappear during the course of the disease, being consistent with the dynamic behavior of erosion over time. Based on these striking results it seems reasonable to suggest that the new-bone formation process in SpA could be associated with the resolution of cortical entheseal erosion over time. These results could also be in agreement with the apparent failure of anti-tumor necrosis factor (TNF) therapies to control bone proliferation in SpA; and with the relation of TNF-α, Dickkopf-related protein 1 (Dkk-1) and the regulatory molecule of the Wnt signaling pathway in the bone proliferation in SpA. In the same model, we then proceeded to study the enthesis bursa. Interestingly, the Outcome Measures in Rheumatology Clinical Trials (OMERACT) enthesopathy definition does not include bursa as an elementary entheseal lesion. Nonetheless, bursa was included in 46% of the enthesis studies in a recently systematic literature review, being in agreement with the concept of “synovio-entheseal complex” that includes the link between enthesitis and osteitis in SpA. It has been clarified in recent data that there is not only a close functional integration of the enthesis with the neighboring bone, but also a connection between enthesitis and synovitis. Therefore, we tried to assess the prevalence and relevance of the bursa-synovial lesion in SpA. Our findings showed a significant increase of Achilles bursa presence and thickness in SpA patients compared to controls (healthy/mechanical controls and RA controls). These results raise awareness to the need to improve the enthesopathy ultrasonographic definition. In the final work of this thesis, we have explored new perspectives, not previously reported, about construct validity of enthesis ultrasound as a possible activity outcome in SpA. We performed a longitudinal Achilles enthesis ultrasound study in patients with early SpA. Achilles ultrasound examinations were performed at baseline, six- and twelve-month time periods and compared with clinical outcome measures collected at basal visit. Our results showed that basal erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are higher in patients with Doppler signal in enthesis, and even that higher basal ESR, CRP and Ankylosing Spondylitis Disease Activity Score (ASDAS) predicted a higher Doppler signal (an ultrasound alteration accepted as representative of inflammation) six months later. Patients with very high disease activity assessed by ASDAS (>3.5) at baseline had significantly higher Achilles total ultrasound score verified at the same time; and ASDAS <1.3 predicted no Doppler signal at six and twelve months. This seems to represent a connection between classical biomarkers and clinical outcomes associated with SpA activity and Doppler signal, not only at the same time, but also for the following months. Remarkably, patients with inactive disease (ASDAS < 1.3) at baseline had no Doppler signal at six and twelve months. These findings reinforce the potential use of ultrasound related techniques for disease progression assessment and prognosis purposes. Intriguingly, Ankylosing Spondylitis Disease Activity Index (BASDAI) didn’t show significant differences between different cut-offs concerning ultrasound lesions or Doppler signal, while verified with ASDAS. These results seem to indicate that ASDAS reflects better than BASDAI what happens in the enthesis. The work herein discussed clearly shows the potential utility of ultrasound in enthesis assessment in SpA patients, and can be important for the development of ultrasound activity and structural damage scores for diagnosis and monitoring purposes. Therefore, local promotion of this technique constitutes a medical intervention that is worth being tested in SpA patients for diagnosis, monitoring and prognosis purposes.

Characterization of earth-based mortars for rammed earth repair

All over the world, many earth buildings are deteriorating due to lack of maintenance and repair. Repairs on rammed earth walls are mainly done with mortars, by rendering application; however, often the repair is inadequate, resorting to the use of incompatible materials, including cement-based mortars. It has been observed that such interventions, in walls that until that day only had presented natural ageing issues, created new problems, much more dangerous for the building than the previous ones, causing serious deficiencies in this type of construction. One of the problems is that the detachment of the new cement-based mortar rendering only occurs after some time but, until that occurrence, degradations develop in the wall itself. When the render detaches, instead of needing only a new render, the surface has to be repaired in depth, with a repair mortar. Consequently, it has been stablished that the renders, and particularly repair mortars, should have physical, mechanical and chemical properties similar to those of the rammed earth walls. This article intends to contribute to a better knowledge of earth-based mortars used to repair the surface of rammed earth walls. The studied mortars are based on four types of earth: three of them were collected from non-deteriorated parts of walls of unstabilized rammed earth buildings located in Alentejo region, south of Portugal; the fourth is a commercial earth, consisting mainly of clay. Other components were also used, particularly: sand to control shrinkage; binders stabilizers such as dry hydrated air-lime, natural hydraulic lime, Portland cement and natural cement; as well as natural vegetal fibers (hemp fibers). The experimental analysis of the mortars in the fresh state consisted in determining the consistency by flow table and the bulk density. In the hardened state, the tests made it possible to evaluate the following properties: linear and volumetric shrinkage; capillary water absorption; drying capacity; dynamic modulus of elasticity; flexural and compressive strength.

Earthen plasters based on illitic soils from Barrocal region of Algarve: contributions for building performance and sustainability

Clayish earth-based mortars can be considered eco-efficient products for indoor plastering since they can contribute to improve important aspects of building performance and sustainability. Apart from being products with low embodied energy when compared to other types of mortars used for interior plastering, mainly due to the use raw clay as natural binder, earth-based plasters may give a significant contribution for health and comfort of inhabitants. Due to high hygroscopicity of clay minerals, earth-based mortars present a high adsorption and desorption capacity, particularly when compared to other type of mortars for interior plastering. This capacity allows earth-based plasters to act as a moisture buffer, balancing the relative humidity of the indoor environment and, simultaneously, acting as a passive removal material, improving air quality. Therefore, earth-based plasters may also passively promote the energy efficiency of buildings, since they may contribute to decreasing the needs of mechanical ventilation and air conditioning. This study is part of an ongoing research regarding earth-based plasters and focuses on mortars specifically formulated with soils extracted from Portuguese ‘Barrocal’ region, in Algarve sedimentary basin. This region presents high potential for interior plastering due to regional geomorphology, that promote the occurrence of illitic soils characterized by a high adsorption capacity and low expansibility. More specifically, this study aims to assess how clayish earth and sand ratio of mortars formulation can influence the physical and mechanical properties of plasters. For this assessment four mortars were formulated with different volumetric proportions of clayish earth and siliceous sand. The results from the physical and mechanical characterization confirmed the significantly low linear shrinkage of all the four mortars, as well as their extraordinary adsorption-desorption capacity. These results presented a positive correlation with mortars´ clayish earth content and are consistent with the mineralogical analysis, that confirmed illite as the prevalent clay mineral in the clayish earth used for this study. Regarding mechanical resistance, although the promising results of the adhesion test, the flexural and compressive strength results suggest that the mechanical resistance of these mortars should be slightly improved. Considering the present results the mortars mechanical resistance improvement may be achieved through the formulation of mortars with higher clayish earth content, or alternatively, through the addition of natural fibers to mortars formulation, very common in this type of mortars. Both those options will be investigated in future research.

Analysis of a controversial decision process: the case of the pumped hydro storage power plant Atdorf in Germany

Based on the report for the unit “Métodos Interactivos de Participação e Decisão A” (Interactive methods of participation and decision A), coordinated by Prof. Lia Maldonado Teles de Vasconcelos and Prof. Nuno Miguel Ribeiro Videira Costa. This unit was provided for the PhD Program in Technology Assessment in 2015/2016.

Damage propagation in composite Materials meso-mechanical models

Composite materials have a complex behavior, which is difficult to predict under different types of loads. In the course of this dissertation a methodology was developed to predict failure and damage propagation of composite material specimens. This methodology uses finite element numerical models created with Ansys and Matlab softwares. The methodology is able to perform an incremental-iterative analysis, which increases, gradually, the load applied to the specimen. Several structural failure phenomena are considered, such as fiber and/or matrix failure, delamination or shear plasticity. Failure criteria based on element stresses were implemented and a procedure to reduce the stiffness of the failed elements was prepared. The material used in this dissertation consist of a spread tow carbon fabric with a 0°/90° arrangement and the main numerical model analyzed is a 26-plies specimen under compression loads. Numerical results were compared with the results of specimens tested experimentally, whose mechanical properties are unknown, knowing only the geometry of the specimen. The material properties of the numerical model were adjusted in the course of this dissertation, in order to find the lowest difference between the numerical and experimental results with an error lower than 5% (it was performed the numerical model identification based on the experimental results).

A type system for value-dependent information flow analysis

Information systems are widespread and used by anyone with computing devices as well as corporations and governments. It is often the case that security leaks are introduced during the development of an application. Reasons for these security bugs are multiple but among them one can easily identify that it is very hard to define and enforce relevant security policies in modern software. This is because modern applications often rely on container sharing and multi-tenancy where, for instance, data can be stored in the same physical space but is logically mapped into different security compartments or data structures. In turn, these security compartments, to which data is classified into in security policies, can also be dynamic and depend on runtime data. In this thesis we introduce and develop the novel notion of dependent information flow types, and focus on the problem of ensuring data confidentiality in data-centric software. Dependent information flow types fit within the standard framework of dependent type theory, but, unlike usual dependent types, crucially allow the security level of a type, rather than just the structural data type itself, to depend on runtime values. Our dependent function and dependent sum information flow types provide a direct, natural and elegant way to express and enforce fine grained security policies on programs. Namely programs that manipulate structured data types in which the security level of a structure field may depend on values dynamically stored in other fields The main contribution of this work is an efficient analysis that allows programmers to verify, during the development phase, whether programs have information leaks, that is, it verifies whether programs protect the confidentiality of the information they manipulate. As such, we also implemented a prototype typechecker that can be found at http://ctp.di.fct.unl.pt/DIFTprototype/.