Sulfonate and acetyl derivatives of the vasopressin antagonist tolvaptan: reactivity towards DNA and 2’-deoxynucleosides

Tolvaptan is an orally effective nonpeptide arginine vasopressin (AVP) V2-receptor antagonist, used in the treatment of clinically significant euvolemic and hypervolemic hyponatremia. Nonetheless, Tolvaptan was reported to be associated with a high risk of liver injury, prompting the U. S. Food and Drug Administration (FDA) to issue a black box label warning. Whereas the mechanisms of tolvaptan-induced liver injury remain to be elucidated, preliminary results obtained in cytochrome P450 (CYP)-deficient liver cell lines have shown evidence of tolvaptaninduced DNA damage without the need for Phase I metabolism. Taking into consideration that Phase II sulfonation and/or acetylation of tolvaptan’s secondary alcohol are metabolically plausible events, the ultimate goal of this thesis was to study the reactivity of these putative Phase II metabolites towards DNA. Indeed, the covalent adducts thus afforded could explain the tolvaptan-induced DNA damage observed in CYP-deficient liver cell lines. Since the identification of DNA adducts involves the hydrolysis to 2’-deoxynucleobase adducts followed by liquid chromatography coupled with mass spectrometry (LC-ESI-MS) in comparison with adducts standards, the first step of this thesis was the preparation of tolvaptan-DNA adduct standards. Two distinct strategies were followed: 1) biomimetic, involving the reaction of the metabolic plausible sulfate and acetate tolvaptan metabolites with 2’-deoxynucleobases; 2) non-biomimetic, involving a palladium-catalyzed coupling reaction. For the biomimetic approach, the two metabolic plausible Phase II metabolites were first prepared: 1) 5’-O-sulfonate-tolvaptan was prepared in 89 % yield upon tolvaptan sulfonation using sulfur trioxide-pyridine complex; 2) 5’-O-acetyl-tolvaptan was prepared in 80% following tolvaptan acetylation with acetic anhydride and trimethylamine. The reaction of 5’-O-sulfonate-tolvaptan and 5’-O-acetyl-tolvaptan with 2’-deoxynucleobases (and nucleophilic amino acids) were subsequently undergone, with the ultimate goal of preparing adduct standards. However, only the product stemming from the elimination of sulfoxy and acetoxy groups was detected, upon LC-ESI-MS analysis of these reaction mixtures. For the non-biomimetic strategy, the derivative 5’-chloro-tolvaptan was prepared upon reaction of tolvaptan with thionyl chloride, and subsequently coupled with 3′,5′-O-bis(tertbutyldimethylsilyl)-2′-deoxyguanosine, under palladium catalysis. Following deprotection, the LC-ESI-HRMS analysis of the reaction mixture allowed the identification of two covalent adducts, one non-depurinating and one depurinating. The reaction of 5’-O-sulfonate-tolvaptan and 5’-O-acetyl-tolvaptan with DNA were also undergone. However, the LC-ESI-HRMS analysis of the hydrolysates obtained following enzymatic or thermic hydrolysis did not allow the identification of any tolvaptan-DNA adduct. This result suggests that the DNA damage observed in hepatic cell lines does not stem from the formation of DNA adducts with the metabolic plausible Phase II metabolites, 5’-O-sulfonate-tolvaptan and 5’-O-acetyltolvaptan

Farms in a box : how a new e-commerce solution is disrupting the purchasing of fresh produce online

Box scheme is a service where consumers order fresh fruits and vegetables boxes online and that is delivered directly by producers. This service has emerged as a rapidly growing alternative to conventional grocery deliver systems. However, consumers are not attracted to buy fresh fruits and vegetables online, due to the variable quality and sensory nature of this category. Thus, this study aims to identify the factors that influence consumers’ willingness to purchase fresh fruits and/or vegetables box by e-commerce and to identify which box’s characteristics contributes to the overall customers’ satisfaction leading to repurchase intentions. These factors depend entirely on the online environment and not on the sensory experience of the product itself. Therefore, through an online questionnaire, a sample of 284 participants was collected and used to analyze the results, using confirmatory factor analysis and multiple linear regressions. The results obtained revealed that perceived quality, perceived healthiness, perceived convenience and information availability positively affect consumers’ willingness to purchase the boxes online. Hence, when consumers are willing to order a box online they create expectations for a desire outcome influencing their overall satisfaction with it. In addition, the degree of overall customers’ satisfaction, which has a positive impact on their repurchase intentions, is affected by the level of satisfaction with certain box characteristics, such as flexibility of box composition, freshness of the products and service precision.Os cabazes de frutas e legumes frescos são encomendados online pelo consumidor e são entregues diretamente pelos produtores. Este serviço apareceu como uma alternativa em rápida expansão aos convencionais sistemas de entrega dos supermercados. Contudo, a compra de frutas e legumes frescos online não atrai os consumidores, devido à qualidade variável e à natureza sensorial desta categoria. Assim, o objetivo deste estudo é identificar os fatores que influenciam a disposição dos consumidores de comprar um cabaz de frutas e/ou legumes frescos através do comércio eletrônico e identificar as características do cabaz que contribuem para a satisfação geral dos clientes, levando a intenções de recompra. Esses fatores dependem inteiramente do ambiente online e não da experiência sensorial do próprio produto. Assim, através de um questionário online foi recolhida uma amostra de 284 participantes que foi usada para a análise dos resultados, feita através de análise fatorial confirmatória e regressões lineares múltiplas. Os resultados obtidos revelaram que a qualidade percebida, saúde percebida, conveniência percebida e disponibilidade de informação afetam positivamente a disposição dos consumidores de comprar os cabazes online. Consequentemente, os consumidores estando dispostos a encomendar um cabaz online criam expectativas do resultado desejado influenciando a sua satisfação geral com o mesmo. Além disso, o grau de satisfação geral dos clientes, que tem um impacto positivo nas intenções de recompra dos mesmos, é afetado pelo nível de satisfação com certas características do cabaz, tais como a flexibilidade da composição do cabaz, a frescura dos produtos e a precisão do serviço

LPBlocks: a block-based language for linear programming

Dissertação de mestrado em Informatics EngineeringLinear programming is a mathematical optimization technique used in numerous fields including mathematics, economics, and computer science, with numerous industrial contexts, including solving optimization problems such as planning routes, allocating resources, and creating schedules. As a result of its wide breadth of applications, a considerable amount of its user base lacks programming knowledge and experience and thus often resorts to using graphical software such as Microsoft Excel. However, despite its popularity amongst less technical users, the methodologies used by these tools are often ad-hoc and prone to errors. Block-based languages have been successfully used to aid novice programmers and even children in programming. Thus, we created a block-based programming language termed LPBlocks that allows users to create linear programming models using data contained in spreadsheets. This language guides the users to write syntactically and semantically correct programs and thus aids them in a way that current languages do not. We have also implemented a web application where users can define linear programming models, reactively see their mathematical representation and execute them to obtain the optimization values for the variables defined by the users. To assess the applicability of LPBlocks we used it to successfully express numerous and varied linear programming problems. Furthermore, we designed and ran a qualitative empirical study to understand the experience our tool and language brings to users from various backgrounds. Although we see differences amongst the users, most of them were able to model several problems using LPBlocks.Programação linear é um conjunto de técnicas de otimização matemática utilizada em várias áreas estas incluem matemática, economia, ciências da computação e usos em contextos industriais, incluindo planear rotas, alocar recursos e planear horários. Como resulta das suas aplicações variadas uma grande quantidade dos seus utilizadores não possuem conhecimentos de programação e por isso utilizam software gráfico como o Microsoft Excel. Apesar da sua popularidade este software utiliza metodologias ad-hoc e propicias a erros. As linguagem de programação por blocos tem surgido nos últimos anos com o intuito de ajudar programadores iniciantes, tendo mesmo aplicações no ensino de crianças. Sendo assim nos criamos uma linguagem de programação pro blocos que utiliza dados contidos em folhas de calculo para criar modelos de programação linear chamada LPBlocks. Esta linguagem guia utilizadores na criação de modelos semanticamente e sintaticamente corretos. Para avaliar a validade de LPBlocks nos implementamos vários problemas utilizando a mesma. Posteriormente implementamos esta linguagem e utilizamo-la num estudo com utilizadores de vários níveis de experiência. Depois utilizamos a informação recolhida durante o estudo para avaliar LPBlocks e propor melhorias

Aerosols and Bacteria From Hand Washing and Drying in Indoor Air

Effective hand drying is an important part of hand hygiene that can reduce the risk of infectious disease transmission through cross-contamination of surfaces by wet hands. However, hand drying methods may also cause aerosolisation of pathogenic microorganisms if they are present in washed hands. This study investigated experimentally the impact of washing hands and different hand drying methods on the concentration and size distribution of aerosols and bacteria in indoor air. In this experiment, aerosol and bacteria concentrations were measured in indoor air while volunteers rinsed their hands with water or washed with soap and water prior to drying them with paper towels or jet air dryers. Results showed that the concentration of aerosols and bacteria in air increased with people walking in the room and washing hands, with a further increase during the hand drying process. The concentration of aerosols decreased with particle size, with maximum concentrations after drying hands of 6.63 × 106 ± 6.49 × 105 and 2.28 × 104 ± 9.72 × 103 particles m−3 for sizes 0.3 to &lt;0.5 and ≥5.0 μm, respectively. The concentration of bacteria in indoor air after drying hands increased to a maximum of 3.81 × 102 ± 1.48 × 102 CFU m−3 (jet air dryers) and 4.50 × 102 ± 4.35 × 101 CFU m−3 (paper towels). This study indicates that the increase of aerosols and bacteria in air after drying hands with jet air dryers or paper towels are comparable and not statistically different from concentrations associated with walking and washing hands in the same environment. This work can support the development of hand hygiene practices and guidelines for public washrooms.</p

Disclosing CCBE1 role in Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiovascular diseases (CVD) are the leading cause of death worldwide. Within CVDs, myocardial infarction (MI) is associated with a massive and irreversible loss of cardiomyocytes (CM). An in-depth comprehension of key cellular mechanisms and molecules involved in cardiogenesis is fundamental to improve cardiac therapies by exposing novel therapeutic targets. CCBE1, a collagen and calcium-EGF biding domain 1 protein, was identified to be expressed in mouse heart precursors. Mutations in CCBE1 have been associated with Hennekam syndrome, which is characterized by abnormal lymphatic system and congenital heart defects. However, the CCBE1 functional role in cardiac specification is still unknown. Therefore, the main aim of this thesis was to unveil CCBE1 role in CM and Endothelial cells (EC) specification. For this purpose, a modified hiPSC line displaying the CRISPR interference technology (CRISPRi) was used to selectively knockdown (KD) CCBE1 gene expression along CM and EC differentiation. We showed that CCBE1 downregulation did not affect hiPSCs growth, morphology and stemness. Nonetheless, a significant reduction on gene expression of cardiac troponin T2 gene (TNNT2) and lower gene expression ratios of cardiac troponin I isoforms (TNNI3:TNNI1) and myosin heavy chains (MYH7:MYH6) were detected in CMs derived from CRISPRi-CCBE1 KD cell line at day 15. Ultrastructural changes were also observed in this condition, CMs presented lower sarcomere length and alignment, indicating a more immature state. In contrast, EC differentiation was not affected by CCBE1 KD, with no impact on EC morphology or gene expression levels. Therefore, CCBE1 seems to have a key role on CM specification and maturation. Moreover, we successfully selected hiPSC clonal populations with higher level of CCBE1 KD for future studies. This work may contribute with new insights towards the development of CCBE1-mediated therapeutic strategies for cardiac regenerative medicine

Upgrading of the model to determine the economic and financial feasibility of a hotel

This work project encompasses the upgrading of an already existing model to determine a hotel´s economic and financial feasibility, by adding some figures that give more information to the hotel managers and, therefore, can guide them in their strategic decisions. This upgrading was conducted to adapt the model to the changes that are going on in the hospitality industry such as, the growing importance of digital technology, that is changing the balance of power towards the consumer side and the change in the consumer behaviours and expectations since the new generations value more the local experiences and plan their trips using online sources. The model will be based in the principles of the Uniform System of Accounts to the Lodging Industry (USALI), published by the American Hotel Lodging Educational Institute. The first edition was published in 1926 with the objective of enable the hotels to recording, classifying and publishing the hotel´s internal results in a format that is equal among all hotels in the world. This will allow the results to be compared worldwide and, also, contribute to the creation of reliable indicators in the industry, crucial for all players in this market. USALI was the first successful accounting system and, nowadays, is the most accepted worldwide

The impact of ADAS in the insurance world

Internship Report presented as the partial requirement for obtaining a Master's degree in Data Science and Advanced Analytics, specialization in Business AnalyticsThe automobile industry in insurance companies has been increasingly becoming more competitive and innovative, seeking not only to increase its portfolio, but especially to attract customers with fewer claims, thus increasing profitability and reducing risk. Cars have evolved their equipment, making them more “intelligent”, in order to ensure a more comfortable driving and to warn of dangers on the road. One of the reasons vehicles are becoming more intelligent are devices called ADAS (Advanced Driver-Assistance Systems). This project aims to understand the impact that these devices have on the severity and frequency, through predictive models. With this, it will be possible to delineate discounts for insurance policies of vehicles that have this equipment, while simultaneously verifying if these vehicles are less prone to accidents

Rowing training assessment using smart sensors

Technology is evolving, and the appearance of new smart sensors is increasing. The use of sensors to improve performance during sports activities is getting more relevant than ever. Thus, a system capable of obtaining real-time force metrics of a rowing machine's footplate and seat was developed. The force sensors on the footplate, connected to the Shimmer3 will obtain the heels and toes force information sent through a Bluetooth connection to the developed mobile application. In this application, the rower can understand during training how to improve his rowing technique. Additionally, it is possible to access the history information with more metrics and the evolution of all training sessions. Finally, this system can also detect the force exercised on the seat and the distribution of the centre of force, helping the athlete understand which side it tends to lean in all phases of the rowing stroke.Com o desenvolvimento da tecnologia e aparecimento de novos sensores inteligentes, é cada vez mais imergente a utilização dos mesmos para melhorar a performance de atletas durante a atividade desportiva. Deste modo, foi desenvolvido um sistema capaz de obter em tempo real métricas de força do pau de voga e slide de uma máquina de remo. Os sensores de força no pau de voga, ligados ao Shimmer3 vão obter a informação de força dos calcanhares e ponta do pé que serão enviados através de uma ligação Bluetooth para a aplicação móvel desenvolvida. Nesta aplicação, o remador consegue perceber durante o treino como melhorar a sua técnica de remada. Adicionalmente, ainda é possível aceder a um histórico de informação onde se encontram mais métricas e a evolução de todos os treinos. Por fim, este sistema também consegue detetar a força exercida no slide bem como a distribuição do centro de força ajudando o atleta a perceber para que lado é exercida mais pressão em todas a fases da remada

Manipulation of carbon nanoparticles in composites with electric fields for improved electrical properties

Die Dispersion elektrisch leitfähiger Nanopartikel in einer Polymermatrix ermöglicht die Herstellung einer nützlichen Materialklasse: elektrisch leitfähige Polymerkomposite. Die gezielte Einstellung der elektrischen Leitfähigkeit des leitfähigen Komposits ist jedoch nicht trivial, da viele Material- und Verarbeitungsparameter die Partikel-Netzwerkstruktur im Komposit beeinflussen die letztlich für die elektrische Leitfähigkeit ausschlaggebend ist. In dieser Arbeit werden die elektrischen Eigenschaften von Epoxid-Nanokompositen mit Kohlenstoff-Nanopartikeln als leitfähigem Füllstoff für verschiedene Verarbeitungsbedingungen untersucht. Im Unterschied zu einem klassischen Formgebungsverfahren für Epoxidmaterialien wird die Anwendung von elektrischen Feldern während des Aushärtungsprozesses als zusätzlicher Prozessparameter benutzt. Elektrische Felder werden während des Aushärtens an die Nanokomposite angelegt, wodurch die elektrischen Eigenschaften des Endmaterials durch Induktion von Polarisations- und Dipol-Wechselwirkungen zwischen den leitenden Partikeln (Dielektrophorese) beeinflusst werden. Diese führen zu einer neuen, und im Hinblick auf die elektrische Leitfähigkeit vorteilhaften Mikro- und Nanostruktur des leitfähigen Netzwerks. Dieses Phänomen wird für ein System von einwandigen Kohlenstoff-Nanoröhren in einer Epoxidharzmatrix im Hinblick auf den Einfluss verschiedener Verarbeitungsparameter auf die elektrische Leitfähigkeit des Systems untersucht. Darauf aufbauend wird ein skalierbares Verfahren auf der Grundlage des Harzinjektionsverfahrens zur Herstellung von Nanokompositen unter elektrischen Feldern entwickelt. Für ein besseres Verständnis der Mechanismen, die der Wirkung elektrischer Felder auf die Rotation und die Verschaltung von SWCNTs werden die eigenen Ergebnisse mit Simulationen kooperierender Wissenschaftler der Anáhuac Mayab Universit¨1t (Merida, Mexiko) und das Imperial College London (Vereinigtes Königreich) verglichen

Antimicrobial susceptibility and molecular characterization of resistance mechanisms of Enterococcus spp. from the intestinal microbiota of food producing animals

O trato intestinal dos mamíferos constitui um reservatório natural de Enterococcus, bactérias oportunistas, ubiquitárias, frequentemente associadas a infeções nosocomiais multirresistentes em humanos, sendo Enterococcus faecium e Enterococcus faecalis as espécies mais importantes. Neste estudo foram identificadas as espécies de Enterococcus predominantes no conteúdo cecal de bovinos e suínos através de técnicas moleculares, tendo sido avaliados os perfis de suscetibilidade antimicrobiana das estirpes de E. faecium e E. faecalis pelos métodos de agar difusão e microdiluição, e identificados determinantes de resistência por PCR e Whole Genome Sequencing. Os suínos constituem um reservatório de estirpes multirresistentes de E. faecium e E. faecalis. Enteroccocus spp. resistentes a antibióticos de importância crítica, designadamente daptomicina e linezolid, foram identificados em intestino de bovinos e suínos, tendo sido detectados os determinantes de resistência ao linezolid (optrA e poxtA). Estes resultados enfatizam a importância de monitorizar a resistência antimicrobiana em bactérias de origem animal; ABSTRACT: The intestinal tract of mammals is a natural reservoir of Enterococcus, opportunistic and ubiquitous bacteria, frequently associated with multidrug resistant nosocomial infections in humans, Enterococcus faecium and Enterococcus faecalis being the most important species. In the present study, the prevalent species of Enterococcus in the cecum of cattle and pigs were identified through molecular techniques, the antimicrobial susceptibility profiles of E. faecium and E. faecalis strains were assessed by agar diffusion and microdilution methods, and resistant determinants were identified through PCR and Whole Genome Sequencing. Pigs are a reservoir of multidrug resistant E. faecium and E. faecalis strains. Enterococcus spp. resistant to critically important antibiotics, namely daptomycin and linezolid, were found colonizing bovine and swine gut, and the corresponding linezolid resistance determinants were identified (optrA and poxtA). These results highlight the importance of monitoring antimicrobial resistance mechanisms in bacteria from animals