Virulent Shigella flexneri subverts the host innate immune response through manipulation of antimicrobial peptide gene expression

Antimicrobial factors are efficient defense components of the innate immunity, playing a crucial role in the intestinal homeostasis and protection against pathogens. In this study, we report that upon infection of polarized human intestinal cells in vitro, virulent Shigella flexneri suppress transcription of several genes encoding antimicrobial cationic peptides, particularly the human β-defensin hBD-3, which we show to be especially active against S. flexneri. This is an example of targeted survival strategy. We also identify the MxiE bacterial regulator, which controls a regulon encompassing a set of virulence plasmid-encoded effectors injected into host cells and regulating innate signaling, as being responsible for this dedicated regulatory process. In vivo, in a model of human intestinal xenotransplant, we confirm at the transcriptional and translational level, the presence of a dedicated MxiE-dependent system allowing S. flexneri to suppress expression of antimicrobial cationic peptides and promoting its deeper progression toward intestinal crypts. We demonstrate that this system is also able to down-regulate additional innate immunity genes, such as the chemokine CCL20 gene, leading to compromised recruitment of dendritic cells to the lamina propria of infected tissues. Thus, S. flexneri has developed a dedicated strategy to weaken the innate immunity to manage its survival and colonization ability in the intestine

Electronic Devices for the Combination of Electrically Controlled Drug Release, Electrostimulation, and Optogenetic Stimulation for Nerve Tissue Regeneration

[ES] La capacidad de las células madre para proliferar formando distintas células especializadas les otorga la potencialidad de servir de base para terapias efectivas para patologías cuyo tratamiento era inimaginable hasta hace apenas dos décadas. Sin embargo, esta capacidad se encuentra mediada por estímulos fisiológicos, químicos, y eléctricos, específicos y complejos, que dificultan su traslación a la rutina clínica. Por ello, las células madre representan un campo de estudio en el que se invierten amplios esfuerzos por parte de la comunidad científica. En el ámbito de la regeneración nerviosa, para modular su desarrollo y diferenciación el tratamiento farmacológico, la electroestimulación, y la estimulación optogenética son técnicas que están consiguiendo prometedores resultados. Es por ello por lo que en la presente tesis se ha desarrollado un conjunto de sistemas electrónicos para permitir la aplicación combinada de estas técnicas in vitro, con perspectiva a su aplicación in vivo. Hemos diseñado una novedosa tecnología para la liberación eléctricamente controlada de fármacos. Esta tecnología está basada en nanopartículas de sílice mesoporosa y puertas moleculares de bipiridina-heparina. Las puertas moleculares son electroquímicamente reactivas, y encierran los fármacos en el interior de las nanopartículas, liberándolos ante un estímulo eléctrico. Hemos caracterizado esta tecnología, y la hemos validado mediante la liberación controlada de rodamina en cultivos celulares de HeLa. Para la combinación de liberación controlada de fármacos y electroestimulación hemos desarrollado dispositivos que permiten aplicar los estímulos eléctricos de forma configurable desde una interfaz gráfica de usuario. Además, hemos diseñado un módulo de expansión que permite multiplexar las señales eléctricas a diferentes cultivos celulares. Además, hemos diseñado un dispositivo de estimulación optogenética. Este tipo de estimulación consiste en la modificación genética de las células para que sean sensibles a la radiación lumínica de determinada longitud de onda. En el ámbito de la regeneración de tejido mediante células precursoras neurales, es de interés poder inducir ondas de calcio, favoreciendo su diferenciación en neuronas y la formación de circuitos sinápticos. El dispositivo diseñado permite obtener imágenes en tiempo real mediante microscopía confocal de las respuestas transitorias de las células al ser irradiadas. El dispositivo se ha validado irradiando neuronas modificadas con luz pulsada de 100 ms. También hemos diseñado un dispositivo electrónico complementario de medida de irradiancia con el doble fin de permitir la calibración del equipo de irradiancia y medir la irradiancia en tiempo real durante los experimentos in vitro. Los resultados del uso de los bioactuadores en procesos complejos y dinámicos, como la regeneración de tejido nervioso, son limitados en lazo abierto. Uno de los principales aspectos analizados es el desarrollo de biosensores que permitiesen la cuantización de ciertas biomoléculas para ajustar la estimulación suministrada en tiempo real. Por ejemplo, la segregación de serotonina es una respuesta identificada en la elongación de células precursoras neurales, pero hay otras biomoléculas de interés para la implementación de un control en lazo cerrado. Entre las tecnologías en el estado del arte, los biosensores basados en transistores de efecto de campo (FET) funcionalizados con aptámeros son realmente prometedores para esta aplicación. Sin embargo, esta tecnología no permitía la medición simultánea de más de una biomolécula objetivo en un volumen reducido debido a las interferencias entre los distintos FETs, cuyos terminales se encuentran inmersos en la solución. Por ello, hemos desarrollado instrumentación electrónica capaz de medir simultáneamente varios de estos biosensores, y la hemos validado mediante la medición simultánea de pH y la detección preliminar de serotonina y glutamato.[CA] La capacitat de les cèl·lules mare per a proliferar formant diferents cèl·lules especialitzades els atorga la potencialitat de servir de base per a teràpies efectives per a patologies el tractament de les quals era inimaginable fins fa a penes dues dècades. No obstant això, aquesta capacitat es troba mediada per estímuls fisiològics, químics, i elèctrics, específics i complexos, que dificulten la seua translació a la rutina clínica. Per això, les cèl·lules mare representen un camp d'estudi en el qual s'inverteixen amplis esforços per part de la comunitat científica. En l'àmbit de la regeneració nerviosa, per a modular el seu desenvolupament i diferenciació el tractament farmacològic, l'electroestimulació, i l'estimulació optogenética són tècniques que estan aconseguint prometedors resultats. És per això que en la present tesi s'ha desenvolupat un conjunt de sistemes electrònics per a permetre l'aplicació combinada d'aquestes tècniques in vitro, amb perspectiva a la seua aplicació in vivo. Hem dissenyat una nova tecnologia per a l'alliberament elèctricament controlat de fàrmacs. Aquesta tecnologia està basada en nanopartícules de sílice mesoporosa i portes moleculars de bipiridina-heparina. Les portes moleculars són electroquímicament reactives, i tanquen els fàrmacs a l'interior de les nanopartícules, alliberant-los davant un estímul elèctric. Hem caracteritzat aquesta tecnologia, i l'hem validada mitjançant l'alliberament controlat de rodamina en cultius cel·lulars de HeLa. Per a la combinació d'alliberament controlat de fàrmacs i electroestimulació hem desenvolupat dispositius que permeten aplicar els estímuls elèctrics de manera configurable des d'una interfície gràfica d'usuari. A més, hem dissenyat un mòdul d'expansió que permet multiplexar els senyals elèctrics a diferents cultius cel·lulars. A més, hem dissenyat un dispositiu d'estimulació optogenètica. Aquest tipus d'estimulació consisteix en la modificació genètica de les cèl·lules perquè siguen sensibles a la radiació lumínica de determinada longitud d'ona. En l'àmbit de la regeneració de teixit mitjançant cèl·lules precursores neurals, és d'interés poder induir ones de calci, afavorint la seua diferenciació en neurones i la formació de circuits sinàptics. El dispositiu dissenyat permet obtindré imatges en temps real mitjançant microscòpia confocal de les respostes transitòries de les cèl·lules en ser irradiades. El dispositiu s'ha validat irradiant neurones modificades amb llum polsada de 100 ms. També hem dissenyat un dispositiu electrònic complementari de mesura d'irradiància amb el doble fi de permetre el calibratge de l'equip d'irradiància i mesurar la irradiància en temps real durant els experiments in vitro. Els resultats de l'ús dels bioactuadors en processos complexos i dinàmics, com la regeneració de teixit nerviós, són limitats en llaç obert. Un dels principals aspectes analitzats és el desenvolupament de biosensors que permeteren la quantització de certes biomolècules per a ajustar l'estimulació subministrada en temps real. Per exemple, la segregació de serotonina és una resposta identificada amb l'elongació de les cèl·lules precursores neurals, però hi ha altres biomolècules d'interés per a la implementació d'un control en llaç tancat. Entre les tecnologies en l'estat de l'art, els biosensors basats en transistors d'efecte de camp (FET) funcionalitzats amb aptàmers són realment prometedors per a aquesta aplicació. No obstant això, aquesta tecnologia no permetia el mesurament simultani de més d'una biomolècula objectiu en un volum reduït a causa de les interferències entre els diferents FETs, els terminals dels quals es troben immersos en la solució. Per això, hem desenvolupat instrumentació electrònica capaç de mesurar simultàniament diversos d'aquests biosensors i els hem validat amb mesurament simultani del pH i la detecció preliminar de serotonina i glutamat.[EN] The stem cells' ability to proliferate to form different specialized cells gives them the potential to serve as the basis for effective therapies for pathologies whose treatment was unimaginable until just two decades ago. However, this capacity is mediated by specific and complex physiological, chemical, and electrical stimuli that complicate their translation to clinical routine. For this reason, stem cells represent a field of study in which the scientific community is investing a great deal of effort. In the field of nerve regeneration, to modulate their development and differentiation, pharmacological treatment, electrostimulation, and optogenetic stimulation are techniques that are achieving promising results. For this reason, we have developed a set of electronic systems to allow the combined application of these techniques in vitro, with a view to their application in vivo. We have designed a novel technology for the electrically controlled release of drugs. This technology is based on mesoporous silica nanoparticles and bipyridine-heparin molecular gates. The molecular gates are electrochemically reactive and entrap the drugs inside the nanoparticles, releasing them upon electrical stimulus. We have characterized this technology and validated it by controlled release of rhodamine in HeLa cell cultures. For combining electrostimulation and controlled drug release we have developed devices that allow applying the different electrical stimuli in a configurable way from a graphical user interface. In addition, we have designed an expansion module that allows multiplexing electrical signals to different cell cultures. In addition, we have designed an optogenetic stimulation device. This type of stimulation consists of genetically modifying cells to make them sensitive to light radiation of a specific wavelength. In tissue regeneration using neural precursor cells, it is interesting to be able to induce calcium waves, favoring the cell differentiation into neurons and the formation of synaptic circuits. The designed device enable the obtention of real-time images through confocal microscopy of the transient responses of cells upon irradiation. The device has been validated by irradiating modified neurons with 100 ms pulsed light stimulation. We have also designed a complementary electronic irradiance measurement device to allow calibration of the irradiator equipment and measuring irradiance in real time during in vitro experiments. The results of using bioactuators in complex and dynamic processes, such as nerve tissue regeneration, are limited in an open loop. One of the main aspects analyzed is the development of biosensors that would allow quantifying of specific biomolecules to adjust the stimulation provided in real time. For instance, serotonin secretion is an identified response of neural precursor cells elongation, among other biomolecules of interest for the implementation of a closed-loop control. Among the state-of-the-art technologies, biosensors based on field effect transistors (FETs) functionalized with aptamers are promising for this application. However, this technology did not allow the simultaneous measurement of more than one target biomolecule in a small volume due to interferences between the different FETs, whose terminals are immersed in the solution. This is why we have developed electronic instrumentation capable of simultaneously measuring several of these biosensors, and we have validated it with the simultaneous pH measurement and the preliminary detection of serotonin and glutamate.Monreal Trigo, J. (2023). Electronic Devices for the Combination of Electrically Controlled Drug Release, Electrostimulation, and Optogenetic Stimulation for Nerve Tissue Regeneration [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/19384

Development of an Active Finger Motion Simulator: With In-Vitro Assessments of Tendon Loads and Joint Kinematics

Musculoskeletal injuries of the finger far outnumber those of other joints, and while in-vitro motion simulators are useful for studying joint biomechanics and evaluating surgical repairs, considerably less simulator development has been reported for the finger compared to other joints. Replication of active musculoskeletal movement during in-vitro testing has been shown to be more representative of in-vivo motion patterns, and closed-loop motion controllers are the current state-of-the-art for in-vitro kinematics studies. However, an in-vitro motion simulator with closed-loop tendon load control and simultaneous tendon excursion control has not yet been reported for the finger. This thesis outlines the design and development of an active motion simulator for the study of finger joint kinematics, as well as forces and excursions of the flexor/extensor tendons. Performance of the system was verified in terms of tendon load control accuracy and motion repeatability, before conducting two cadaveric studies. An in-vitro study on the effects of wrist position and distal extensor tendon rupture verified that the new simulator produced expected finger kinematics and tendon loads. With the new simulator validated, its high sensitivity in measuring tendon loads was leveraged to observe the effects of A2 and A4 pulley excision and subsequent surgical repair. The thesis culminates with a summary of sample size analysis and statistical effect sizes that will aid future in-vitro finger studies with this simulator

Advanced Techniques for Search-Based Program Repair

Debugging and repairing software defects costs the global economy hundreds of billions of dollars annually, and accounts for as much as 50% of programmers' time. To tackle the burgeoning expense of repair, researchers have proposed the use of novel techniques to automatically localise and repair such defects. Collectively, these techniques are referred to as automated program repair. Despite promising, early results, recent studies have demonstrated that existing automated program repair techniques are considerably less effective than previously believed. Current approaches are limited either in terms of the number and kinds of bugs they can fix, the size of patches they can produce, or the programs to which they can be applied. To become economically viable, automated program repair needs to overcome all of these limitations. Search-based repair is the only approach to program repair which may be applied to any bug or program, without assuming the existence of formal specifications. Despite its generality, current search-based techniques are restricted; they are either efficient, or capable of fixing multiple-line bugs---no existing technique is both. Furthermore, most techniques rely on the assumption that the material necessary to craft a repair already exists within the faulty program. By using existing code to craft repairs, the size of the search space is vastly reduced, compared to generating code from scratch. However, recent results, which show that almost all repairs generated by a number of search-based techniques can be explained as deletion, lead us to question whether this assumption is valid. In this thesis, we identify the challenges facing search-based program repair, and demonstrate ways of tackling them. We explore if and how the knowledge of candidate patch evaluations can be used to locate the source of bugs. We use software repository mining techniques to discover the form of a better repair model capable of addressing a greater number of bugs. We conduct a theoretical and empirical analysis of existing search algorithms for repair, before demonstrating a more effective alternative, inspired by greedy algorithms. To ensure reproducibility, we propose and use a methodology for conducting high-quality automated program research. Finally, we assess our progress towards solving the challenges of search-based program repair, and reflect on the future of the field

Synthesis and characteristics of polyarylene ether sulfones

A method utilizing potassium carbonate/dimethyl acetamide, as base and solvent respectively, was used for the synthesis of several homopolymers and copolymers derived from various bisphenols. It is demonstrated that this method deviates from simple second order kinetics; this deviation being due to the heterogeneous nature of the reaction. Also, it is shown that a liquid induced crystallization process can improve the solvent resistance of these polymers. Finally, a Monte Carlo simulation of the triad distribution of monomers in nonequilibrium copolycondensation is discussed

Modeling and Simulation of Polymerization Processes

This reprint is a compilation of nine papers published in Processes, in a Special Issue on “Modeling and Simulation of Polymerization Processes”. It aimed to address both new findings on basic topics and the modeling of the emerging aspects of product design and polymerization processes. It provides a nice view of the state of the art with regard to the modeling and simulation of polymerization processes. The use of well-established methods (e.g., the method of moments) and relatively more recent modeling approaches (e.g., Monte Carlo stochastic modeling) to describe polymerization processes of long-standing interest in industry (e.g., rubber emulsion polymerization) to polymerization systems of more modern interest (e.g., RDRP and plastic pyrolysis processes) are comprehensively covered in the papers contained in this reprint

MAPPING THE ANKLE JOINTS PASSIVE MOTION AND MEASURING THE EFFECT OF INJURY ON LIGAMENT CONSTRAINT AT THE ANKLE JOINT

Lateral ankle sprain is one of the most common sports injuries, and having a better understanding of how ligament constraint changes after injury can assist in accurately diagnosing ankle laxity, prevention, and recovery. The objective was to map out the range of motion of the ankle under constant torque loading, and to examine the effect of a lateral ankle sprain level 2 and 3. Nine cadaveric ankles were run through a range of motion where a 6 degree of freedom load cell was used to collect force and torque loads and an Optotrak scanner collected the position of the tibia, talus, and calcaneus. The ankle saw more motion in abduction and inversion than adduction and eversion in the talocrural joint, and the ankle joint complex. The subtalar joint saw no change in inversion or eversion, or adduction, but there was less than four degrees of motion in adduction while the foot was plantarflexed. In general the foot was insensitive to varying torque levels with or without injury except in the talocrural joint where inversion and eversion saw a three to four degree difference after a sprain level 3. It was observed that when ABAD torques were applied the ankle was free to move in INEV, but when INEV torques were applied there was a constraint that limited the motion of the ankle in the ABAD direction. The results from this study show the range of motion of the ankle intact, and with a level 2 and level 3 sprain in the subtalar joint, the talocrural joint, and the ankle joint complex. With knowledge of the ankle's envelope of motion and quantitative assessment of the Taylor tilt test could be created, as well during a total ankle replacement different mid-flexion assessments could be made to create better outcomes for patients

Scalable deep learning for bug detection

The application of machine learning (ML) and natural language processing (NLP) methods for creating software engineering (SE) tools is a recent emerging trend. A crucial early decision is how to model software’s vocabulary. Unlike in natural language, software developers are free to create any identifiers they like, and can make them arbitrarily complex resulting in an immense out of vocabulary problem. This fundamental fact prohibits training of Neural models on large-scale software corpora. This thesis aimed on addressing this problem. As an initial step we studied the most common ways for vocabulary reduction previously considered in the software engineering literature and found that they are not enough to obtain a vocabulary of manageable size. Instead this goal was reached by using an adaptation of the Byte-Pair Encoding (BPE) algorithm, which produces an open-vocabulary neural language model (NLM). Experiments on large corpora show that the resulting NLM outperforms other LMs both in perplexity and code completion performance for several programming languages. It continues by showing that the improvement in language modelling transfers to downstream SE tasks by finding that the BPE NLMs are more effective in highlighting buggy code than previous LMs. Driven by this finding and from recent advances in NLP it also investigates the idea of transferring language model representations to program repair systems. Program repair is an important but difficult software engineering problem. One way to achieve a “sweet spot” of low false positive rates, while maintaining high enough recall to be usable, is to focus on repairing classes of simple bugs, such as bugs with single statement fixes, or that match a small set of bug templates. However, it is very difficult to estimate the recall of repair techniques based on templates or based on repairing simple bugs, as there are no datasets about how often the associated bugs occur in code. To fill this gap, the thesis contributes a large dataset of single statement Java bug-fix changes annotated by whether they match any of a set of 16 bug templates along with a methodology for mining similar datasets. These specific patterns were selected with the criteria that they appear often in open-source Java code and relate to those used by mutation and pattern-based repair tools. They also aim at extracting bugs that compile both before and after repair as such can be quite tedious to manually spot, yet their fixes are simple. These mined bugs are quite frequent appearing about every 2000 lines of code and that their fixes are very often already present in the code satisfying the popular plastic surgery hypothesis. Furthermore, it introduces a hypothesis that contextual embeddings offer potential modelling advantages that are specifically suited for modelling source code due to its nature. Contextual embeddings are common in natural language processing but have not been previously applied in software engineering. As such another contribution is the introduction a new set of deep contextualized word representations for computer programs based on the ELMo (embeddings from language models) framework of Peters et al (2018). It is shown that even a low-dimensional embedding trained on a relatively small corpus of programs can improve a state-of-the-art machine learning system for bug detection of single statement fixes. The systems were evaluated on the DeepBugs dataset of synthetic bugs, a new synthetic test dataset, and a small dataset of real JavaScript bugs. Lastly, the final contribution is the first steps at answering whether neural bug-finding is useful in practice by performing an evaluation study over a small set of real bugs

Transport in complex systems : a lattice Boltzmann approach

Celem niniejszej pracy jest zbadanie możliwości efektywnego modelowania procesów transportu w złożonych systemach z zakresu dynamiki płynów za pomocą metody siatkowej Boltzmanna (LBM). Złożoność systemu została potraktowana wieloaspektowo i konkretne układy, które poddano analizie pokrywały szeroki zakres zagadnień fizycznych, m.in. przepływy wielofazowe, hemodynamikę oraz turbulencje. We wszystkich przypadkach szczególna uwaga została zwrócona na aspekty numeryczne — dokładność używanych modeli, jak również szybkość z jaką pozwalają one uzyskać zadowalające rozwiązanie. W ramach pracy rozwinięty został pakiet oprogramowania Sailfish, będący otwarta implementacja metody siatkowej Boltzmanna na procesory kart graficznych (GPU). Po analizie szybkości jego działania, walidacji oraz omówieniu założeń projektowych, pakiet ten został użyty do symulacji trzech typów przepływów. Pierwszym z nich były przepływy typu Brethertona/Taylora w dwu- i trójwymiarowych geometriach, do symulacji których zastosowano model energii swobodnej. Analiza otrzymanych wyników pokazała dobra zgodność z danymi dostępnymi w literaturze, zarówno eksperymentalnymi, jak i otrzymanymi za pomocą innych metod numerycznych. Drugim badanym problemem były przepływy krwi w realistycznych geometriach tętnic dostarczających krew do ludzkiego mózgu. Wyniki symulacji zostały dokładnie porównane z rozwiązaniem otrzymanym metoda objętości skończonych z wykorzystaniem pakietu OpenFOAM, przyspieszonego komercyjna biblioteka pozwalająca na wykonywanie obliczeń na GPU. Otrzymano dobra zgodność między badanymi metodami oraz pokazano, że metoda siatkowa Boltzmanna pozwala na wykonywanie symulacji do ok. 20 razy szybciej. Trzecim przeanalizowanym zagadnieniem były turbulentne przepływy w prostych geometriach. Po zwalidowaniu wszystkich zaimplementowanych modeli relaksacji na przypadku wiru Kidy, zbadano przepływy w pustym kanale oraz w obecności przeszkód. Do symulacji wykorzystano zarówno siatki zapewniające pełną rozdzielczość aż do skal Kolmogorova, jak i siatki o mniejszej rozdzielczości. Również w tym kontekście pokazano dobrą zgodność wyników otrzymanych metodą siatkową Boltzmanna z wynikami innych symulacji oraz badaniami eksperymentalnymi. Pokazano również, że implementacja LBM w pakiecie Sailfish zapewnia większą stabilność obliczeń niż ta opisana w literaturze dla tych samych przepływów i modeli relaksacji

Advanced Applications of Rapid Prototyping Technology in Modern Engineering

Rapid prototyping (RP) technology has been widely known and appreciated due to its flexible and customized manufacturing capabilities. The widely studied RP techniques include stereolithography apparatus (SLA), selective laser sintering (SLS), three-dimensional printing (3DP), fused deposition modeling (FDM), 3D plotting, solid ground curing (SGC), multiphase jet solidification (MJS), laminated object manufacturing (LOM). Different techniques are associated with different materials and/or processing principles and thus are devoted to specific applications. RP technology has no longer been only for prototype building rather has been extended for real industrial manufacturing solutions. Today, the RP technology has contributed to almost all engineering areas that include mechanical, materials, industrial, aerospace, electrical and most recently biomedical engineering. This book aims to present the advanced development of RP technologies in various engineering areas as the solutions to the real world engineering problems