Browsing by Issue Date, starting with "2025-02"
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- Uma Introdução à Filosofia da MatemáticaPublication . Castro, Eduardo Jorge de SousaEste livro é um manual para um primeiro curso de Filosofia da Matemática. Pode ser usado ao nível de uma graduação ou de uma pós -graduação, de Filosofia ou de Matemática. Restantes académicos e investigadores, bem como professores do ensino não -universitário, podem também encontrar aqui uma referência e uma orientação para o seu trabalho e investigação. O livro apenas pressupõe conhecimentos de matemática e de filosofia, de nível pré -universitário.
- Characterization of Elite Rink Hockey Players, Match and Training DemandsPublication . Ferraz, António Leopoldo Lopes; Travassos, Bruno Filipe Rama; Santos, João Alberto Valente dos; Mendes, Pedro Alexandre DuarteThis work aimed to provide valuable insights into the characteristics of players, training, and game demands in rink hockey, applying the knowledge developed towards a more suitable method for monitoring training and competition to enhance player performance. To achieve this, six studies were conducted with specific objectives: (1) to understand the evolutionary trends in the characterization of players and game performance in men's rink hockey; (2) to characterize the body composition and grip strength of elite male rink hockey players and establish the relationship between ethnicity in body composition and grip strength; (3) to understand the applicability of monitoring systems for physical performance analysis in team sports over the last decade in order to develop knowledge for load monitoring in rink hockey; (4) to understand the dynamics of external and internal load throughout the 2-week training period and competition week of a rink hockey International Championship; (5) to propose a new approach to characterize and classify the physiological and biomechanical specificity of training tasks in relation to elite competition - a quadrant-based classification system; (6) to apply the quadrant-based classification system to characterize and classify load demands, considering the physiological and biomechanical requirements of training and games according to positional dimensions. Overall, the results obtained in the six studies of this thesis promoted a proposal for practical applicability from player profiling to training and game monitoring. Study 1 revealed that the literature on rink hockey is scarce and generally presents a holistic approach to physiological and functional demands, cardiorespiratory adaptations, training characteristics, anthropometry, body composition, conditional profile, game characterization, and injuries in rink hockey. However, most studies are conducted on young athletes, with few studies on elite athletes. Thus, it was essential to conduct research toward characterizing elite athletes and the physical demands of training and competition. Study 2 described that given the characteristics of the sport and its neuromuscular adaptation, elite rink hockey players show a specific anthropometric profile considering percentile values of fat and characteristic grip strength for this sport, with ethnicity, right thigh circumference, and right calf circumference being the variables that best describe fat percentile, while age, distal circumference of the right thigh, and ethnicity better describe grip strength. Considering that monitoring through positional analysis technology in rink hockey is almost non-existent, Study 3 sought to understand how research using indoor positioning systems could contribute to a better understanding of the demands of rink hockey. It was found that most studies have focused on performance analysis, load management, injury development process, and nutrition. Still, in Study 3, it was proposed that integrated analysis of kinematic and mechanical variables should be studied alongside their correlation or association with internal load variables to understand athletes' performance improvement strategies better. In this logic, Study 4 observed in a preparation for an international competition that training demands do not match those of the competition. High-intensity impacts followed by decelerations characterize the metrics that most impact athletes' subjective perception of effort during competition, which are underexplored during training, particularly high-intensity impacts. Therefore, in Studies 5 and 6, a proposal was developed to characterize physiological and biomechanical demands in training and competition situations, providing coaching staff with a methodological tool to optimize microcycle planning. In Study 5, it was found that no exercise category analysed has the physiological and biomechanical demands of the game. While the game is characterized by high physiological and biomechanical demands, most exercises have low physiological and biomechanical demands, except for exercises performed in midcourt and full court, which have high physiological and medium biomechanical demands. Still, in Study 5, no category of exercises with simultaneous high biomechanical and low physiological demands was found. Finally, in study 6, a misalignment between the specific demands of different positions was observed when comparing the game with the training sessions of the microcycle. That is, although the game is characterized by high physiological and biomechanical efforts, advanced players tend to achieve a lower physiological and biomechanical profile than defender-midfielder players. These differences are not considered throughout the microcycle. Finally, this approach used in Studies 5 and 6 allows us to assume that the integrated variables that best characterize the physiological demands in rink hockey are average heart rate, maximum heart rate, and distances skated at high intensity. In turn, those that best integrate biomechanical demands are high-intensity impacts, decelerations, and accelerations. In summary, the results suggest that training does not meet the demands of the game, compromising athletes' appropriate response capacity, particularly considering the game's biomechanical demands. This is because there is a failure to replicate these demands in training in terms of training structure and player individualization, mainly due to the absence of exercises that promote high-intensity impacts associated with decelerations and accelerations. The quadrant-based classification system proposal presented in studies 5 and 6 offers coaching staff a methodology to analyse and adjust training structure and player specificity, thus promoting an environment that better replicates competition demands.
- From production to purification: Towards an integrative process for recombinant pre-miRNA-29b as biopharmaceuticalPublication . Carapito, Ana Rita Mugeiro; Sousa, Fani Pereira de; Martins, Mara Guadalupe Freire; Sponchioni, MattiaRecent advances in RNA research have greatly demonstrated the potential of RNA-based therapies, offering innovative ways to target a variety of diseases with enhanced specificity. Unlike traditional small-molecule drugs, RNA therapeutics like small-interfering RNA and microRNA (miRNA) can precisely regulate gene expression and target specific biological pathways. For instance, recent studies demonstrate that miRNA-29 regulates some pathological routes associated with Alzheimer's disease (AD), a neurodegenerative disorder affecting millions of people around the world. MiRNA-29 plays a crucial role in processes like amyloid-β peptides (Aβ) formation, which contributes to memory loss and neuronal cell damage. Low levels of miRNA-29 are linked to increased production and activity of the enzyme β-secretase (BACE1), which leads to higher production of Aβ and, consequently, β-plaque formation. Given its important functions, restoring or increasing miRNA-29 levels in AD patients can be a promising strategy for AD treatment. Current research is investigating the use of recombinant miRNA-29b precursor (pre-miRNA-29b) to silence BACE1 expression and decrease Aβ levels, aiming to develop novel approaches to slow the progression of AD. Given this, biopharmaceuticals production and subsequent purification constitute an important process that needs to be in full accordance with criteria established by regulatory entities. Regarding RNA production, the standard technique is through chemical synthesis, however this strategy comes with some disadvantages, namely the biomolecules length that can be correctly produced and the low production levels. Recombinant production is the alternative method that is more cost-effective and applicable for large-scale production. Escherichia coli is the most widely used and studied host, nonetheless, Rhodovulum sulfidophilum (R. sulfidophilum) presents interesting characteristics considering nucleic acids production, that comprise the ability to secrete nucleic acids to the extracellular medium, without secreting RNases. The recovery of recombinantly produced nucleic acids from the extracellular medium might be a great advantage for the further downstream processing because the contaminants, such as cell debris and endotoxins, are not present, as usually are in the intracellular samples. The downstream process is the most expensive stage of the whole bioprocess, and usually, several chromatographic steps are required to achieve the intended purity and quality. Given this, there is a high demand for specific and efficient purification strategies. Multimodal chromatography is currently under thorough research as it can lead to the same specificity for target compounds as it is observed for affinity chromatography, without using biological ligands that greatly increase the process cost. Ionic Liquids (ILs) are molten salts that can present this multimodal character when used as ligands immobilized onto the stationary phase, being recently studied for nucleic acids purification. The different moieties of the cation in an IL can allow the exploitation of different types of interactions with the target molecule. Therefore, this Doctoral Thesis explores a promising and alternative recombinant host for the production of pre-miRNA-29b, as well as its purification using newly synthesized resins, aiming for the development of a whole bioprocess. Initially, a DNA vector was designed to produce the target pre-miRNA-29b in R. sulfidophilum. In this study, the impact of the plasmid on bacterial growth was analysed and compared to the non-transformed strain. The transformed strain has shown a global growth about 5 times lower than the non-transformed strain, but this did not impact negatively the target RNA production. An optimization of the extracellular extraction protocol was also conducted during this study, comparing a protocol with ethanol or isopropanol as precipitation agents. The results proven that the protocol using isopropanol as precipitation agent was more efficient reaching a concentration of 0.7 μg of pre-miRNA-29b per liter of medium. After successfully developing an efficient pre-miRNA-29b production system, it became important to develop a purification strategy. For this, four different silica-based Ionic Liquids (SILs) were synthesized and evaluated on their ability for nucleic acids separation. An initial screening of binding and elution conditions with a low molecular weight RNA sample was performed by using both ionic and hydrophobic conditions, to select the more promising support for further purification assays. The support SSi[C3C3NH2Im]Cl was selected, proving to be highly efficient in separating different species of DNA (genomic and plasmidic) from RNA. Then, it became important to test this support regarding its ability for pre-miRNA-29b purification from other types of RNAs. Given that this approach is more challenging due to the high physical and chemical similarity among small RNAs, four different ILs were employed in this stage to act as competition agents aiming to enhance target selectivity. From the four tested ILs, the 1-ethylimidazolium chloride was proven to have a higher impact on the pre-miRNA-29b selectivity improvement, achieving 76% purity. Since the aim is to develop a method suitable for a biopharmaceutical production and purification, it would be needed to achieve higher purity levels than what was verified with SSi[C3C3NH2Im]Cl. Therefore, a commercial multimodal resin, Capto Q ImpRes was chosen to analyse and compare its performance to the previous newly synthesized support. Both intra- and extracellular RNA samples were tested regarding pre-miRNA-29b purification, and a Design of Experiments (DoE) was established for each sample. Screening assays with intracellular RNA sample were made to identify the factors to be implemented in the DoE. Sodium chloride concentration and pH were defined, and it was possible to establish conditions that uncover a balance between the recovery and purity of the target. For intracellular RNA samples, the DoE effectively identified optimal purification parameters, attaining recovery rates up to 73% and purity levels around 78%. However, these two responses shown to be almost inversely proportional. Nonetheless, it was possible to achieve the optimal point with a recovery of 48.21% and purity of 51.15%. On the other hand, purifying extracellular pre-miRNA-29b presented substantial challenges, exhibiting significant inconsistencies in purity and difficulties in identifying the target molecule, probably due to the low concentration of pre-miRNA in the extracellular extract and the difficulty in detection. To search for an alternative that could lead to an improved selectivity for the biopharmaceutical under study, a set of several oligonucleotides were designed to interact with the target. Initially, 13 oligonucleotides were designed to interact through base complementarity with different regions of the target. Each oligonucleotide was linked to a carbon chain (6 or 12 carbons) with an amino group, resulting in 26 different ligands. After data analysis, some ligands showed higher specificity for their target sites, while others demonstrated versatility in recognizing multiple sites. Based on this, 4 oligonucleotides were identified as the most promising for further experimental testing. Overall, in this doctoral thesis, a bioprocess was developed starting with the upstream stage with recombinant production of the pre-miRNA-29b, followed by the downstream stage exploiting different approaches applicable to target purification. The work opens the route for the development of an integrated production and purification process for pre-miRNA-29b, with great potential for large-scale translation.
- Detection of Stealthy Distributed Denial of Service Attacks Using Artificial Intelligence MethodsPublication . Rios, Vinícius de Miranda; Freire, Mário Marques; Magoni, DamienDistributed Denial of Service (DDoS) attacks have been used to disrupt various online activities. The significant traffic volume of these distributed attacks has enabled the identification of signatures and behavior profiles that fostered the development of detection mechanisms for mitigating these attacks. However, as new attack types emerge, such as low-rate Denial of Service (DoS) attacks, new detection mechanisms need to be developed to combat these evolving threats effectively. Many detection mechanisms rely primarily on statistical analysis to identify low-rate DoS attacks in data traffic. However, these methods often exhibit a high rate of false negatives and are only applicable to small-scale data. Artificial intelligence techniques have been widely employed in various fields, including social network analysis and disease monitoring, and have gradually gained prominence in the field of cybersecurity in recent years. This thesis focuses on studying and developing detection mechanisms that exhibit effective performance against two specific types of low-rate DoS attacks: the Reduction of Quality (RoQ) attack and the Slowloris attack. For the RoQ attack, we examine the traffic transmission format to create a similar one, as there is no existing software capable of generating this type of attack traffic on the internet. For the Slowloris attack, we utilized free and open-source software specifically developed for this purpose. Subsequently, we analyze the traffic from both attacks and extract features that can be used by detection mechanisms. In this thesis, two approaches have been developed for classifying and detecting RoQ and Slowloris attacks: one approach is based on the separate use of a set of traditional Machine Learning (ML) algorithms and the second approach is based on fuzzy logic plus one traditional ML algorithm (that previously led to good classification results) and Euclidean distance. For the RoQ attack detection, the first approach uses eleven separate machine learning algorithms, namely K-Nearest Neighbors (K-NN), Multilayer Perceptron Neural Network (MLP), Support Vector Machine (SVM), Multinomial Naive Bayes (MNB), Gaussian Naive Bayes (GNB), Decision Tree (DT), Random Forest (RF), Gradient Boosting (XGB), Logistic Regression (LR), AdaBoost, and Light Gradient Boosting Machine (LGBM), while the second approach consists in our proposed method which combines fuzzy logic, the MLP algorithm, and the Euclidean distance method. For the Slowloris attack detection, the first approach utilizes nine machine learning algorithms, namely KNN, GNB, MLP, SVM, DT, MNB, RF, XGB, and LGBM, while the second approach consists in our proposed method which combines fuzzy logic, the RF algorithm, and the Euclidean distance method. Both approaches utilize previously selected features to classify the data traffic as either attack traffic or legitimate traffic. The obtained results show that some ML algorithms (namely MLP and RF) as well as our approach based on fuzzy logic, one ML algorithm, and Euclidean distance are good candidates to be used to classify RoQ and Slowloris attacks, but the latter approach with a slightly longer runtime for detecting them.