Monitoring the ex-vivo expansion of human mesenchymal stem/stromal cells in xeno-free microcarrier-based reactor systems by MIR spectroscopy

Human mesenchymal stem/stromal cells (MSCs) have received considerable attention in the field of cell-based therapies due to their high differentiation potential and ability to modulate immune responses. However, since these cells can only be isolated in very low quantities, successful realization of these therapies requires MSCs ex-vivo expansion to achieve relevant cell doses. The metabolic activity is one of the parameters often monitored during MSCs cultivation by using expensive multi-analytical methods, some of them time-consuming. The present work evaluates the use of mid-infrared (MIR) spectroscopy, through rapid and economic high-throughput analyses associated to multivariate data analysis, to monitor three different MSCs cultivation runs conducted in spinner flasks, under xeno-free culture conditions, which differ in the type of microcarriers used and the culture feeding strategy applied. After evaluating diverse spectral preprocessing techniques, the optimized partial least square (PLS) regression models based on the MIR spectra to estimate the glucose, lactate and ammonia concentrations yielded high coefficients of determination (R2 ≥ 0.98, ≥0.98, and ≥0.94, respectively) and low prediction errors (RMSECV ≤ 4.7%, ≤4.4% and ≤5.7%, respectively). Besides PLS models valid for specific expansion protocols, a robust model simultaneously valid for the three processes was also built for predicting glucose, lactate and ammonia, yielding a R2 of 0.95, 0.97 and 0.86, and a RMSECV of 0.33, 0.57, and 0.09 mM, respectively. Therefore, MIR spectroscopy combined with multivariate data analysis represents a promising tool for both optimization and control of MSCs expansion processes.

Media Brokerage: Agent-Based SLA Negotiation

Media content personalisation is a major challenge involving viewers as well as media content producer and distributor businesses. The goal is to provide viewers with media items aligned with their interests. Producers and distributors engage in item negotiations to establish the corresponding service level agreements (SLA). In order to address automated partner lookup and item SLA negotiation, this paper proposes the MultiMedia Brokerage (MMB) platform, which is a multiagent system that negotiates SLA regarding media items on behalf of media content producer and distributor businesses. The MMB platform is structured in four service layers: interface, agreement management, business modelling and market. In this context, there are: (i) brokerage SLA (bSLA), which are established between individual businesses and the platform regarding the provision of brokerage services; and (ii) item SLA (iSLA), which are established between producer and distributor businesses about the provision of media items. In particular, this paper describes the negotiation, establishment and enforcement of bSLA and iSLA, which occurs at the agreement and negotiation layers, respectively. The platform adopts a pay-per-use business model where the bSLA define the general conditions that apply to the related iSLA. To illustrate this process, we present a case study describing the negotiation of a bSLA instance and several related iSLA instances. The latter correspond to the negotiation of the Electronic Program Guide (EPG) for a specific end viewer.

Intelligent decision support systems for collaboration in industrial plants

Dissertação apresentada para obtenção do Grau de Doutor em Sistemas de Informação Industriais, Engenharia Electrotécnica, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Biological activity of well defined hydantoin derivatives on efflux pump systems of bacteria and cancer cells

A multi-resistência a antibióticos e medicamentos usados em quimioterapia é um dos grandes problemas com os quais as instituições de saúde se debatem hoje em dia. A acção provocada por bombas de efluxo é uma das suas causas. Estas bombas têm uma importância fundamental, uma vez que, ao expelirem todo o tipo de tóxicos para o exterior das células, também expelem medicamentos, fazendo com que estes não tenham o efeito desejado dentro delas. As bombas de efluxo são transportadores que se encontram nas membranas de todo o tipo de células. Existem dois grandes tipos de bombas de efluxo: as primárias e as secundárias. As primeiras conferem multi-resistência principalmente em células eucariotas, como as células do cancro em humanos, tendo como função a mediação da repulsa de substâncias tóxicas por intermédio da hidrólise de ATP. A primeira a ser descoberta e mais estudada destas bombas foi a ABCB1 que é o gene que codifica a glicoproteína-P (P de permeabilidade). Enquanto as secundárias, que são a maior fonte de multi-resistência em bactérias, promovem a extrusão de substâncias tóxicas através da força motriz de protões. Neste tipo de bombas são conhecidas quatro famílias principais, das quais uma das mais importantes é a superfamília RND, uma vez que inclui a bomba AcrAB-TolC, que é muito importante no metabolismo xenobiótico de bactérias Gramnegativas, nomeadamente a E.coli. Com o objectivo de reverter a multi-resistência, tanto em células eucariotas como procariotas, têm-se desenvolvido estratégias de combate que envolvem a descoberta de substâncias que inibam as bombas de efluxo. Assim sendo, ao longo dos tempos têm sido descobertas variadas substâncias que cumprem este objectivo. É o caso, por exemplo, dos derivados de fluoroquinolonas usados como inibidores de bombas de efluxo em bactérias ou do Tamoxifen, utilizado na terapia de pacientes com cancro da mama. Um dos grupos de substâncias estudados para o desenvolvimento de possíveis compostos que actuem como reversores de multi-resistência são os compostos derivados de hidantoínas. Estes, são conhecidos por possuírem uma grande variedade de propriedades bioquímicas e farmacológicas, sendo portanto usados para tratarem algumas doenças em humanos, como a epilepsia. Nestes, estão englobados compostos com actividade anti-convulsão que constitui a sua grande mais-valia e, dependente da substituição no anel que os constitui, uma grande variedade de outras propriedades farmacológicas como a anti-fungica, a anti-arritmica, a anti-viral, a anti-diabética ou por exemplo a antagonização de determinados receptores, como os da serotonina. Apesar de pouco usados em estudos experimentais para desenvolver substâncias anti-carcinogénicas, existem alguns estudos com este efeito. Objectivos: O presente projecto envolve o estudo de bombas de efluxo primárias e secundárias, em células eucariotas e procariotas, respectivamente. Em bactérias, foram usados quatro modelos experimentais: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, E. coli AG 100 e Salmonella Enteritidis NCTC 13349. Em células de cancro foram usadas, células T de linfoma de rato parentais e células T de linfoma de rato transfectadas com o gene humano MDR-1. O principal objectivo deste estudo foi a pesquisa de novos moduladores de bombas de efluxo presentes em bactérias e células do cancro, tentando assim contribuir para o desenvolvimento de novos agentes farmacológicos que consigam reverter a multi-resistência a medicamentos. Assim sendo foram testados trinta compostos derivados de hidantoínas: SZ-2, SZ-7, LL-9, BS-1, JH-63, MN-3, TD-7k, GG-5k, P3, P7, P10, P11, RW-15b, AD-26, RW-13, AD-29, KF-2, PDPH-3, Mor-1, KK-XV, Thioam-1, JHF-1, JHC-2, JHP-1, Fur-2, GL-1, GL-7, GL-14, GL-16, GL-18. Como forma de atingir estes objectivos, a actividade biológica dos trinta compostos derivados de hidantoínas foi avaliada nas quatro estirpes de bactérias da seguinte forma: foram determinadas as concentrações mínimas inibitórias dos trinta compostos como forma de definir as concentrações em que os compostos seriam utilizados. Os compostos foram posteriormente testadas com um método fluorométrico de acumulação de brometo de etídeo, que é um substrato comum em bombas de efluxo bacterianas, desenvolvido por Viveiros et al. A actividade biológica dos compostos derivados de hidantoínas nas células de cancro foi demonstrada por diferentes métodos. O efeito anti-proliferativo e citotóxico dos trinta compostos foi avaliado nas células T de linfoma de rato transfectadas com o gene humano MDR-1 pelo método de thiazolyl de tetrazólio (MTT). Como o brometo de etídeo também é expelido pelos transportadores ABC, estes compostos foram posteriormente testados com um método fluorométrico de acumulação de brometo de etídeo desenvolvido por Spengler et al nos dois diferentes tipos de células eucariotas. Resultados: A maioria dos compostos derivados de hidantoínas foi eficaz na modulação de bombas de efluxo, nas duas estirpes de bactérias Gram-negativas e nos dois diferentes tipos de células T de linfoma. Em contraste com estes resultados, nas duas estirpes de células Gram-positivas, a maioria dos compostos tiveram pouco efeito na inibição de bombas de efluxo ou até nenhum, em muitos dos casos. De uma maneira geral os melhores compostos nas diferentes estirpes de bactérias foram: Thioam-1, SZ-2, P3, Rw-15b, AD-26, AD-29, GL-18, GL-7, KF-2, SZ-7, MN-3, GL-16 e GL- 14. Foram portanto estes os compostos que provocaram maior acumulação de brometo de etídeo, inibindo assim com maior eficácia as bombas de efluxo. No presente estudo, a maioria dos compostos conseguiu inibir a resistência provocada pela bomba de efluxo ABCB1, tanto nas células parentais bem como nas células que sobre-expressam esta bomba, causando a acumulação de brometo de etídeo dentro das células. As células que sobreexpressam a bomba ABCB1 foram posteriormente testadas com citometria de fluxo que é a técnica padrão para pesquisa de inibidores de bombas de efluxo. Os compostos que foram mais efectivos na inibição da bomba ABCB1, causando assim maior acumulação de brometo de etídeo nas células que sobre-expressam esta bomba foram: PDPH-3, GL-7, KK-XV, AD-29, Thioam-1, SZ-7, KF-2, MN-3, RW-13, LL-9, P3, AD-26, JH-63 e RW- 15b. Este facto não corroborou totalmente os resultados da citometria de fluxo uma vez que os moduladores que provocaram maior inibição da bomba ABCB1 foram o MN-3, JH-63 e o BS-1, sendo que o último não foi seleccionado como um bom composto usando o método fluorométrico de acumulação de brometo de etídeo. Conclusão: Os compostos derivados de hidantoínas testados tiveram maior efeito nas estirpes de bactérias Gram-negativas do que nas Gram-positivas. Relativamente às células eucariotas, as estruturas mais activas apresentam substituintes aromáticos bem como alguns fragmentos aminicos terciários.

Expression of antibodies and retroviralVectors from defined chromosomal sites: strategies towards reliable production systems

Dissertation presented to obtain a Ph.D. degree in Sciences of Engineering and Technology, Cell Technology, at the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

An agent-based evolutionary approach for manufacturing system layout design

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Engenharia Electrotécnica e de Computadores

Intra-Task Device Scheduling for Real-Time Embedded Systems

An ever increasing need for extra functionality in a single embedded system demands for extra Input/Output (I/O) devices, which are usually connected externally and are expensive in terms of energy consumption. To reduce their energy consumption, these devices are equipped with power saving mechanisms. While I/O device scheduling for real-time (RT) systems with such power saving features has been studied in the past, the use of energy resources by these scheduling algorithms may be improved. Technology enhancements in the semiconductor industry have allowed the hardware vendors to reduce the device transition and energy overheads. The decrease in overhead of sleep transitions has opened new opportunities to further reduce the device energy consumption. In this research effort, we propose an intra-task device scheduling algorithm for real-time systems that wakes up a device on demand and reduces its active time while ensuring system schedulability. This intra-task device scheduling algorithm is extended for devices with multiple sleep states to further minimise the overall device energy consumption of the system. The proposed algorithms have less complexity when compared to the conservative inter-task device scheduling algorithms. The system model used relaxes some of the assumptions commonly made in the state-of-the-art that restrict their practical relevance. Apart from the aforementioned advantages, the proposed algorithms are shown to demonstrate the substantial energy savings.

Logic-based schedulability analysis for compositional hard real-time embedded systems

Over the past decades several approaches for schedulability analysis have been proposed for both uni-processor and multi-processor real-time systems. Although different techniques are employed, very little has been put forward in using formal specifications, with the consequent possibility for mis-interpretations or ambiguities in the problem statement. Using a logic based approach to schedulability analysis in the design of hard real-time systems eases the synthesis of correct-by-construction procedures for both static and dynamic verification processes. In this paper we propose a novel approach to schedulability analysis based on a timed temporal logic with time durations. Our approach subsumes classical methods for uni-processor scheduling analysis over compositional resource models by providing the developer with counter-examples, and by ruling out schedules that cause unsafe violations on the system. We also provide an example showing the effectiveness of our proposal.

A framework for memory contention analysis in multi-core platforms

The last decade has witnessed a major shift towards the deployment of embedded applications on multi-core platforms. However, real-time applications have not been able to fully benefit from this transition, as the computational gains offered by multi-cores are often offset by performance degradation due to shared resources, such as main memory. To efficiently use multi-core platforms for real-time systems, it is hence essential to tightly bound the interference when accessing shared resources. Although there has been much recent work in this area, a remaining key problem is to address the diversity of memory arbiters in the analysis to make it applicable to a wide range of systems. This work handles diverse arbiters by proposing a general framework to compute the maximum interference caused by the shared memory bus and its impact on the execution time of the tasks running on the cores, considering different bus arbiters. Our novel approach clearly demarcates the arbiter-dependent and independent stages in the analysis of these upper bounds. The arbiter-dependent phase takes the arbiter and the task memory-traffic pattern as inputs and produces a model of the availability of the bus to a given task. Then, based on the availability of the bus, the arbiter-independent phase determines the worst-case request-release scenario that maximizes the interference experienced by the tasks due to the contention for the bus. We show that the framework addresses the diversity problem by applying it to a memory bus shared by a fixed-priority arbiter, a time-division multiplexing (TDM) arbiter, and an unspecified work-conserving arbiter using applications from the MediaBench test suite. We also experimentally evaluate the quality of the analysis by comparison with a state-of-the-art TDM analysis approach and consistently showing a considerable reduction in maximum interference.

A Multi-DAG Model for Real-Time Parallel Applications with Conditional Execution

The 30th ACM/SIGAPP Symposium On Applied Computing (SAC 2015). 13 to 17, Apr, 2015, Embedded Systems. Salamanca, Spain.

Allocation of Parallel Real-Time Tasks in Distributed Multi-core Architectures supported by an FTT-SE Network

Distributed real-time systems such as automotive applications are becoming larger and more complex, thus, requiring the use of more powerful hardware and software architectures. Furthermore, those distributed applications commonly have stringent real-time constraints. This implies that such applications would gain in flexibility if they were parallelized and distributed over the system. In this paper, we consider the problem of allocating fixed-priority fork-join Parallel/Distributed real-time tasks onto distributed multi-core nodes connected through a Flexible Time Triggered Switched Ethernet network. We analyze the system requirements and present a set of formulations based on a constraint programming approach. Constraint programming allows us to express the relations between variables in the form of constraints. Our approach is guaranteed to find a feasible solution, if one exists, in contrast to other approaches based on heuristics. Furthermore, approaches based on constraint programming have shown to obtain solutions for these type of formulations in reasonable time.

Study of forbidden transitions in atomic systems

A Thesis submitted for the co-tutelle degree of Doctor in Physics at Universidade Nova de Lisboa and Université Pierre et Marie Curie

Task partitioning and priority assignment for distributed hard real-time systems

In this paper, we propose the Distributed using Optimal Priority Assignment (DOPA) heuristic that finds a feasible partitioning and priority assignment for distributed applications based on the linear transactional model. DOPA partitions the tasks and messages in the distributed system, and makes use of the Optimal Priority Assignment (OPA) algorithm known as Audsley’s algorithm, to find the priorities for that partition. The experimental results show how the use of the OPA algorithm increases in average the number of schedulable tasks and messages in a distributed system when compared to the use of Deadline Monotonic (DM) usually favoured in other works. Afterwards, we extend these results to the assignment of Parallel/Distributed applications and present a second heuristic named Parallel-DOPA (P-DOPA). In that case, we show how the partitioning process can be simplified by using the Distributed Stretch Transformation (DST), a parallel transaction transformation algorithm introduced in [1].

P-SOCRATES: A parallel software framework for time-critical many-core systems

Article in Press, Corrected Proof

Gait Analysis of Natural and Artificial Walking Systems

This paper studies periodic gaits of multi-legged locomotion systems based on dynamic models. The purpose is to determine the system performance during walking and the best set of locomotion variables. For that objective the prescribed motion of the robot is completely characterized in terms of several locomotion variables such as gait, duty factor, body height, step length, stroke pitch, foot clearance, legs link lengths, foot-hip offset, body and legs mass and cycle time. In this perspective, we formulate three performance measures of the walking robot namely, the mean absolute energy, the mean power dispersion and the mean power lost in the joint actuators per walking distance. A set of model-based experiments reveals the influence of the locomotion variables in the proposed indices.