129 research outputs found

    Strategies to Improve Antineoplastic Activity of Drugs in Cancer Progression

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    The aim of this Special Issue is to collect reports regarding all the recent strategies, directed at the improvement of antineoplastic activity of drugs in cancer progression, engaging all the expertise needed for the development of new anticancer drugs: medicinal chemistry, pharmacology, molecular biology, and computational and drug delivery studies

    Comparative analysis of metallated phthalocyanines for photodynamic therapy of solid tumors

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    Photodynamic therapy (PDT) comprises the administration of a photosensitizer (PS) and its accumulation within the tumor site, followed by irradiation with light of a specific wavelength. Consequently, singlet oxygen and other reactive oxygen/nitrogen species (ROS/RNS) are produced from bioavailable oxygen at the tumor’s microenvironment and are responsible for the tumor’s eradication. PDT works effectively in certain types of cancer, but poorly in tumors that reside in internal organs and organ structures such as the pancreas and biliary tree. Moreover, adverse effect such as skin phototoxicity is a major obstacle to more widespread clinical applicability. To that end, we encapsulated a second-generation of PS into liposomal carriers that are targeted to the tumor interstitium after intravenous administration. Therefore, this doctoral thesis describes essentially the evaluation and comparison between lipophilic metallated-phthalocyanines (ZnPC and AlPC) encapsulated in interstitially-targeted liposomes (ITLs), and their corresponding hydrophilic derivatives (ZnPCS4 and AlPCS4). To do so, we initiated the doctoral research through our first study by performing an attritional assessment in vitro using A431 cells as a template for tumor cells with a dysfunctional P53 tumor suppressor gene and epidermal growth factor receptor (EGFR) overexpression. As a methodology for our investigations, we have first assessed the dark toxicity as a function of PS concentration using the water-soluble tetrazolium salt (WST-1) and sulforhodamine B dyes as an indication of the cell viability. Using the same principle, we then drew the LC50 values for each PS through PDT at 671 nm and a light exposure of 15 J/cm2 following 1 hour of PS exposure. We continued our research looking into a time-dependent uptake and intracellular distribution of the PS, and we finalized our first study with the assessment of the mode of cell death as well as the cell cycle arrest at 24 hours after PDT. Through this research we observed that, in the absence of illumination, AlPC and ZnPC in ITLs were not toxic to cells up to a 1.5 μM PS concentration and exposure for up to 72 h, but for AlPCS4 and ZnPCS4, the dark toxicity was at 5 μM and at 2.5 μM, respectively. However, PDT of cells photosensitized with ZnPC, AlPC, and AlPCS4 yielded LC50 values of 0.13 μM, 0.04 μM, and 0.81 μM, respectively (24 hours post-PDT based on sulforhodamine B assay). The uptake of all PSs was observed as early as 1 min after PS addition to cells and increased in amplitude during a 2-h incubation period. ZnPCS4 did not induce notable phototoxicity, which was echoed in the mode of cell death and cell cycle arrest data. However, AlPCS4 induced considerable necrosis in addition to apoptosis, whereby most of the cell death had already manifested as early as 2 h after PDT. Cell death signaling coincided with a reduction in cells in the G0/G1 phase (ZnPC, AlPC, AlPCS4) and cell cycle arrest in the S-phase (ZnPC, AlPC, AlPCS4) and G2 phase (ZnPC and AlPC). With the intention of validating our previous research, we have moved forward with the investigations through a second study by using our comparative model with the four metallated-phthalocyanines in a human cholangiocarcinoma cell line and tumor-comprising cells (endothelial cells, fibroblasts, and macrophages), as a representation of the tumor’s microenvironment. In addition to all parameters assessed in the previous study, we went one step further and evaluated the systemic toxicity of each PS in zebrafish and in chicken embryos, and while using BALB/c nude mice as our in vivo model, we researched for signs of skin phototoxicity. A pilot study on PDT efficacy was also performed in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts. The key findings were that photodynamically active PSs (all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells. In addition, PSs in study did not induced any notable systemic toxicity in zebrafish and chicken embryos. However, ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum containing PSs did not exert any detectable sign of cutaneous phototoxicity. Last but not least, ITL-delivered ZnPC and AlPC are equally effective in their tumor-killing capacity in human tumor breast cancer xenografts, and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis. In summary, the research on the applications of ZnPCS4 for oncological PDT will be discontinued in our group as it failed the attrition step regarding phototoxicity and it showed alarming signs of cutaneous phototoxicity. It is therefore concluded that AlPC and its derivative AlPCS4 are the least toxic and most effective PSs to employ with respect to ITLs as part of the comprehensive tumor targeting and PS delivery platform.A terapia fotodinâmica (do inglês PDT) baseia-se essencialmente na administração de um fotossensibilizador e da sua acumulação no local do tumor, seguido de irradiação com luz de comprimento de onda específico. Consequentemente, o oxigénio singleto e outras espécies reativas de oxigénio/nitrogénio (ROS/RNS) são produzidos a partir de oxigénio biodisponível no microambiente do tumor, e são os principais responsáveis pela erradicação do tumor por meio de três mecanismos distintos: 1) morte celular por apoptose/necrose/autofagia; 2) rutura microvascular do tumor mediado por trombose; e 3) uma resposta anti-tumoral do sistema imunitário. PDT funciona de forma eficaz em certos tipos de cancro, mas menos eficaz em tumores que se alojam em órgãos internos e estruturas de órgãos, como por exemplo: o pâncreas e a árvore biliar. Além disso, efeitos adversos como a fototoxicidade da pele, são um grande obstáculo para uma ampla aplicabilidade no setor clínico. Os pacientes precisam de residir em ambientes escuros durante semanas após a terapia, para se protegerem da luz solar, o que é anti-ético nos casos em que os pacientes têm apenas alguns meses de vida. Nesse sentido, o objetivo da presente tese consiste na investigação de fotonanomedicamentos mais adequados para estes pacientes. Para esse fim, foram encapsulados fotossensibilizadores de segunda geração em transportadores lipossomais que são direcionados ao interstício do tumor após administração intravenosa. Os ftalocianine metalados, como o ftalocianine de zinco lipofílico (ZnPC) e o ftalocianine de alumínio (AlPC), bem como seus derivados tetrassulfonados hidrofílicos (ZnPCS4 e AlPCS4), atestam todos os requisitos clínicos para uso em PDT. No entanto, até o momento não houve comparação direta para determinar qual desses fotossensibilizadores é o mais fototóxico para as células tumorais e, portanto, merece mais desenvolvimento pré-clínico e clínico. Esses estudos serão conduzidos de forma a que permita a seleção do fotossensibilizador ideal para o desenvolvimento de uma terceira e quarta geração de fotossensibilizadores (por exemplo, co-encapsulamento de inibidores de vias de sobrevivência de células tumorais com fotossensibilizadores de segunda geração). A presente tese descreve a avaliação e a comparação entre os ftalocianines metalados (ZnPC e AlPC) encapsulados em lipossomas com alvo para o interstício tumoral (ITLs), e os seus equivalentes hidrofílicos (ZnPCS4 e AlPCS4). Para isso, iniciamos a investigação através de uma avaliação in vitro usando células A431 como modelo para células tumorais, com uma disfunção no gene supressor tumoral P53 e superexpressão do receptor de factor de crescimento epidermal (EGFR). Como metodologia numa primeira abordagem foi avaliada a toxicidade no escuro (sem luz de ativação para a PDT) em função da concentração de fotossensibilizadores, utilizando os corantes WST-1 e sulforrodamina B como indicação da viabilidade celular. Seguiu-se o registo da captura celular em detrimento do tempo de exposição com os fotossensibilizadores, e também da sua distribuição a nível intracelular. Ambos os parâmetros foram determinados através da técnica de citometria de fluxo e de microscopia confocal, usando a fluorescência intrínseca dos fotossensibilizadores. Os valores de LC50 foram então estabelecidos para cada PS a um cumprimento de onda de 671 nm e uma exposição radiante de 15 J/cm2, após 1 hora de exposição com os fotossensibilizadores. Finalizamos o nosso primeiro estudo com a avaliação da morte celular em função do tempo pós-PDT, e também com a análise do ciclo celular 24 horas após a PDT. Através desta investigação observamos que, na ausência de iluminação, AlPC e ZnPC em ITLs não foram tóxicos para as células até uma concentração de 1,5 μM (exposição por até 72 h). No entanto, AlPCS4 e ZnPCS4 demonstraram toxicidade no escuro na ordem dos 5 μM e dos 2,5 μM, respetivamente. A absorção de todos os fotossensibilizadores foi observada tão cedo quanto 1 min após a adição de fotossensibilizadores às células, e aumentou em amplitude durante um período de incubação de 2 h. Porém, após 60 min de incubação com os fotossensibilizadores, todo o espaço não nuclear da célula foi fotossensibilizado, com a acumulação dos fotossensibilizadores em múltiplas estruturas subcelulares, especialmente no caso de AlPC e AlPCS4. A PDT de células fotossensibilizadas com ZnPC, AlPC e AlPCS4 foi capaz de produzir valores de LC50 na ordem dos 0,13 μM, 0,04 μM e 0,81 μM, respetivamente (24 horas após PDT, com base no ensaios com sulforrodamina B). O ZnPCS4 não foi capaz de induzir fototoxicidade, o que foi igualmente observado nos resultados obtidos na análise da morte celular e também do ciclo celular. Nas 4 h após PDT, a morte celular resumiu-se principalmente à apoptose para ZnPC e AlPC, que foi gradualmente aumentada em células fotossensibilizadas com AlPC durante 8 h. Por sua vez, as células tratadas com ZnPC recuperaram nas 8 h após PDT em comparação com 4 h pós-PDT. O AlPCS4 foi capaz de induzir a morte celular de forma significativa por necrose para além da apoptose, sendo que a maior parte da morte celular já se havia manifestado 2 h após a PDT. Durante o período de 8 h, a morte celular por via necrótica que era inicialmente a mais predominante, mas foi perdendo para a morte celular apoptótica tardia. A sinalização de morte celular coincidiu com redução de células na fase G0/G1 (ZnPC, AlPC, AlPCS4) e com a redução da atividade celular na fase S (ZnPC, AlPC, AlPCS4) e fase G2 (ZnPC e AlPC) do ciclo celular. A interrupção do ciclo celular foi mais profunda em células que foram incubadas com AlPC e submetidas a PDT. Com a intenção de validar resultados prévios, foram delineadas experiências utilizando o nosso modelo comparativo com os quatro ftalocianines metalados numa linhagem representativa da população celular num tumor (células endoteliais, fibroblastos e macrófagos), e também utilizando células de colangiocarcinoma humano como representante de células cancerígenas. Para além de todos os parâmetros avaliados no estudo anterior, optou-se por dar um passo em frente na investigação, e foi avaliada também a toxicidade sistémica de cada fotossensibilizador em embriões de peixe-zebra (Danio rerio) e galinha (Gallus gallus domesticus). Além disso, determinou-se o risco de fototoxicidade da pele usando murganhos (Mus musculus) BALB/c nude como modelo in vivo. Um estudo piloto sobre a eficácia da PDT também foi realizado em murganhos nude BALB/c portadores de xenoenxertos humanos de cancro de mama triplo negativo (MDA-MB-231). Como principais resultados observou-se que todos os fotossensibilizadores ativos (exceto ZnPCS4) foram capazes de fotossensibilizar efetivamente células cancerígenas e células não cancerosas. Além disso, os fotossensibilizadores em estudo não induziram nenhuma toxicidade sistémica significativa em embriões de peixe-zebra e galinha. No entanto, ZnPCS4 e ZnPC em ITLs foram associados à fototoxicidade da pele, enquanto os fotossensibilizadores contendo alumínio não exerceram nenhuma fototoxicidade detetável no tecido cutâneo. Por último, ZnPC e AlPC encapsulados em ITLs são igualmente eficazes em reduzir o volume tumoral de xenoenxertos de cancro da mama humano em murganhos nude BALB/c, e foram superiores a outros fotossensibilizadores fora do grupo dos ftalocianines quando avaliados em detrimento da dose administrada por mol. Em resumo, as investigações envolvendo o ZnPCS4 para terapia fotodinâmica oncológica serão descontinuadas no nosso grupo, já que ZnPCS4 demonstrou-se menos eficaz comparativamente aos restantes fotossensibilizadores em estudo. Por outro lado, ZnPCS4 revelou também sinais alarmantes de fototoxicidade cutânea. Deste modo, através das investigações incluídas na presente tese de doutoramento concluímos que AlPC e o seu derivativo hidrofílico AlPCS4 são os fotossensibilizadores menos tóxicos e mais eficazes. Assim sendo, pretendem-se continuar com estes dois fotossensibilizadores em futuros estudos rumo ao desenvolvimento de uma plataforma integrando os ITLs juntamente com outro agente quimioterápico para o tratamento eficaz de tumores sólidos através da PDT

    Multiscale Transport and Osmotic Tolerance in Liver Cells and Tissues

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    Cryopreservation enables the storage of biological samples for later use while preserving all aspects of biological interest by cooling them to a temperature where chemical reactions are sufficiently slowed. However, there have been considerable challenges in preserving complex tissues and organs due to excessive ice formation, severe thermal stress, chilling, ischemic injury, and the osmotic stress caused by highly viscous cryoprotectants (CPA). To overcome these challenges, mathematical modeling approaches have proven effective in predicting cell and tissue responses to osmotic stress and developing an optimal method for loading and unloading CPA. Predicting optimal cryopreservation protocols requires an accurate estimation of cell volume, solute concentration, and water permeability parameters. A key bottleneck in this process is the requirement of careful measurement of these parameters from the cellular to the tissue scale and the difficulty of studying these in their native three-dimensional (3D) structures: little is known about the detailed responses of individual cells and nuclei in monolayers and tissues to anisosmotic media. Over the course of four projects, my study has mainly used two approaches to overcome these barriers. It focused on real-time monitoring of cellular morphometric parameters using modern four-dimensional imaging techniques and employed mathematical models for solute and water permeability estimation. In the first project, I characterized the osmotic behavior in HepG2 cells, which serve as a model for hepatocytes, and determined the mechanism of osmoregulation within these cells. I illustrate that HepG2 cells are non-ideal osmometers by showing the difference between the expected behavior of cells in anisosmotic environments and by making predictions about their volume regulation mechanisms. Second, I compared cell volume measurement techniques for adherent cell monolayers, which included using a calcein fluorescence quenching technique to investigate the volumetric responses of HepG2 monolayers. My follow-up study uses modern 3D imaging techniques to simultaneously measure real-time cell and nuclear volume changes in adherent cells in an aniosomotic medium, including during the addition and removal of CPA. My results demonstrate that both cells and nuclei regulate their volume in response to osmotic stress. Consequently, cells and nuclear permeability to water (Lp) and CPA (Ps) are inferred during perfusion with anisosmotic and CPA solutions for adherent cell monolayers. Thirdly, I show that osmotic damage is time dependent and that the flavonoid silymarin enhances resistance to osmotic stress and may improve cryosurvival in HepG2 cells. Finally, I extend the 3D imaging technique to track and quantify three dimensional changes in cell and nuclear morphology in response to anisosmotic medium. I then estimate the volume within complex liver tissue, specifically a precision-cut liver slice (PCLS). This method allows the quantification of the expansion and contraction of the whole PCLS during CPA equilibration, as well as the tracking of nuclei and cell volume. By demonstrating the nonideality of liver cells and the complex interplay between cytoplasm and nuclear volumes, we can inform biophysical models, which may have profound implications for our understanding of cell physiology and the mechanism of osmoregulation. Furthermore, the methods described in this study can be adapted to enhance cryopreservation strategies for adherent cells, other complex tissues, and organs. Altogether, this research contributes to the development of a new cryopreservation method for liver cells and tissues and will have a broad impact on the field of tissue transplantation and biomedical research

    Toward Vision-based Control of Heavy-Duty and Long-Reach Robotic Manipulators

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    Heavy-duty mobile machines are an important part of the industry, and they are used for various work tasks in mining, construction, forestry, and agriculture. Many of these machines have heavy-duty, long-reach (HDLR) manipulators attached to them, which are used for work tasks such as drilling, lifting, and grabbing. A robotic manipulator, by definition, is a device used for manipulating materials without direct physical contact by a human operator. HDLR manipulators differ from manipulators of conventional industrial robots in the sense that they are subject to much larger kinematic and non-kinematic errors, which hinder the overall accuracy and repeatability of the robot’s tool center point (TCP). Kinematic errors result from modeling inaccuracies, while non-kinematic errors include structural flexibility and bending, thermal effects, backlash, and sensor resolution. Furthermore, conventional six degrees of freedom (DOF) industrial robots are more general-purpose systems, whereas HDLR manipulators are mostly designed for special (or single) purposes. HDLR manipulators are typically built as lightweight as possible while being able to handle significant load masses. Consequently, they have long reaches and high payload-to-own-weight ratios, which contribute to the increased errors compared to conventional industrial robots. For example, a joint angle measurement error of 0.5◦ associated with a 5-m-long rigid link results in an error of approximately 4.4 cm at the end of the link, with further errors resulting from flexibility and other non-kinematic aspects. The target TCP positioning accuracy for HDLR manipulators is in the sub-centimeter range, which is very difficult to achieve in practical systems. These challenges have somewhat delayed the automation of HDLR manipulators, while conventional industrial robots have long been commercially available. This is also attributed to the fact that machines with HDLR manipulators have much lower production volumes, and the work tasks are more non-repetitive in nature compared to conventional industrial robots in factories. Sensors are a key requirement in order to achieve automated operations and eventually full autonomy. For example, humans mostly rely on their visual perception in work tasks, while the collected information is processed in the brain. Much like humans, autonomous machines also require both sensing and intelligent processing of the collected sensor data. This dissertation investigates new visual sensing solutions for HDLR manipulators, which are striving toward increased automation levels in various work tasks. The focus is on visual perception and generic 6 DOF TCP pose estimation of HDLR manipulators in unknown (or unstructured) environments. Methods for increasing the robustness and reliability of visual perception systems are examined by exploiting sensor redundancy and data fusion. Vision-aided control using targetless, motion-based local calibration between an HDLR manipulator and a visual sensor is also proposed to improve the absolute positioning accuracy of the TCP despite the kinematic and non-kinematic errors present in the system. It is experimentally shown that a sub-centimeter TCP positioning accuracy was reliably achieved in the tested cases using a developed trajectory-matching-based method. Overall, this compendium thesis includes four publications and one unpublished manuscript related to these topics. Two main research problems, inspired by the industry, are considered and investigated in the presented publications. The outcome of this thesis provides insight into possible applications and benefits of advanced visual perception systems for HDLR manipulators in dynamic, unstructured environments. The main contribution is related to achieving sub-centimeter TCP positioning accuracy for an HDLR manipulator using a low-cost camera. The numerous challenges and complexities related to HDLR manipulators and visual sensing are also highlighted and discussed

    Multi-Robot Systems: Challenges, Trends and Applications

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    This book is a printed edition of the Special Issue entitled “Multi-Robot Systems: Challenges, Trends, and Applications” that was published in Applied Sciences. This Special Issue collected seventeen high-quality papers that discuss the main challenges of multi-robot systems, present the trends to address these issues, and report various relevant applications. Some of the topics addressed by these papers are robot swarms, mission planning, robot teaming, machine learning, immersive technologies, search and rescue, and social robotics

    EG-ICE 2021 Workshop on Intelligent Computing in Engineering

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    The 28th EG-ICE International Workshop 2021 brings together international experts working at the interface between advanced computing and modern engineering challenges. Many engineering tasks require open-world resolutions to support multi-actor collaboration, coping with approximate models, providing effective engineer-computer interaction, search in multi-dimensional solution spaces, accommodating uncertainty, including specialist domain knowledge, performing sensor-data interpretation and dealing with incomplete knowledge. While results from computer science provide much initial support for resolution, adaptation is unavoidable and most importantly, feedback from addressing engineering challenges drives fundamental computer-science research. Competence and knowledge transfer goes both ways

    Indoor Positioning and Navigation

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    In recent years, rapid development in robotics, mobile, and communication technologies has encouraged many studies in the field of localization and navigation in indoor environments. An accurate localization system that can operate in an indoor environment has considerable practical value, because it can be built into autonomous mobile systems or a personal navigation system on a smartphone for guiding people through airports, shopping malls, museums and other public institutions, etc. Such a system would be particularly useful for blind people. Modern smartphones are equipped with numerous sensors (such as inertial sensors, cameras, and barometers) and communication modules (such as WiFi, Bluetooth, NFC, LTE/5G, and UWB capabilities), which enable the implementation of various localization algorithms, namely, visual localization, inertial navigation system, and radio localization. For the mapping of indoor environments and localization of autonomous mobile sysems, LIDAR sensors are also frequently used in addition to smartphone sensors. Visual localization and inertial navigation systems are sensitive to external disturbances; therefore, sensor fusion approaches can be used for the implementation of robust localization algorithms. These have to be optimized in order to be computationally efficient, which is essential for real-time processing and low energy consumption on a smartphone or robot
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