A Graph-Transformation Modelling Framework for Supervisory Control

Formal design methodologies have the potential to accelerate the development and increase the reliability of supervisory controllers designed within industry. One promising design framework which has been shown to do so is known as supervisory control synthesis (SCS). In SCS, instead of manually designing the supervisory controller itself, one designs models of the uncontrolled system and its control requirements. These models are then provided as input to a special synthesis algorithm which uses them to automatically generate a model of the supervisory controller. This outputted model is guaranteed to be correct as long as the models of the uncontrolled system and its control requirements are valid. This accelerates development by removing the need to verify and rectify the model of the supervisory controller. Instead, only the models of the uncontrolled system and its requirements must be validated. To address problems of scale, SCS can be applied in modular fashion, and implemented in hierarchical and decentralized architectures. Despite the large body of research con rming the bene ts of integrating SCS within the development process of supervisory controllers, it has still not yet found widespread application within industry. In the author's opinion, this is partly attributed to the non-user-friendly nature of the automaton-based modelling framework used create the models of the uncontrolled system (and control requirements in even-based SCS). It is believed that in order for SCS to become more accessible to a wider range of non experts, modelling within SCS must be made more intuitive and user-friendly. To improve the usability of SCS, this work illustrates how a graph transformation-based modelling approach can be employed to generate the automaton models required for supervisory control synthesis. Furthermore, it is demonstrated how models of the speci cation can be intuitively represented within our proposed modelling framework for both event- and state-based supervisory control synthesis. Lastly, this thesis assesses the relative advantages brought about by the proposed graph transformation-based modelling framework over the conventional automaton based modelling approach

Predicting Flavonoid UGT Regioselectivity with Graphical Residue Models and Machine Learning.

Machine learning is applied to a challenging and biologically significant protein classification problem: the prediction of flavonoid UGT acceptor regioselectivity from primary protein sequence. Novel indices characterizing graphical models of protein residues are introduced. The indices are compared with existing amino acid indices and found to cluster residues appropriately. A variety of models employing the indices are then investigated by examining their performance when analyzed using nearest neighbor, support vector machine, and Bayesian neural network classifiers. Improvements over nearest neighbor classifications relying on standard alignment similarity scores are reported

Structure and function of Xyloglucan Xylosyltransferases

The plant cell wall is a complex network composed mainly of polysaccharides, the most abundant biopolymers on earth and a rich source of biorenewable materials. Biosynthesis of these plant polysaccharides is poorly understood, largely due to difficulties in the structural characterization of glycosyltransferases and lack of suitable substrates for in vitro analysis. Xyloglucan Xylosyltransferases (XXTs) initiate side-chain extensions from a linear glucan polymer by transferring the xylosyl group from UDP-xylose during xyloglucan biosynthesis. Here, we optimized protein expression and enzymatic activity conditions of XXTs through numerous N- and C-terminal truncations, various E. coli strains, solubility tags, and storage conditions. This procedure was used for protein expression of three XXTs (XXT1, XXT2, and XXT5) and we show that XXT5 is catalytically active in vitro, though at a significantly slower rate compared to XXT1 or XXT2. As no structural information was available for any of the XXTs, we built a homology model of XXT2. This model was used to predict amino acids involved in UDP-xylose binding that were verified through mutagenesis. We subsequently solved the crystal structure of XXT1 without ligands and in complexes with UDP and cellohexaose. XXT1, a homodimer and member of the GT-A fold family of glycosyltransferases, binds UDP analogously to other GT-A fold enzymes. The structures detailed here combined with the properties of mutant XXT1s are consistent with a SNi-like catalytic mechanism. Distinct from other systems is the recognition of cellohexaose by way of an extended cleft. The crystal structure of XXT1 demonstrates that XXT1 alone cannot produce xylosylation patterns observed for native xyloglucans because of steric constraints imposed within the acceptor binding cleft. Homology modeling of XXT2 and XXT5, using the crystal structure of XXT1 as template, reveals a structurally altered cleft in XXT5 that could accommodate a partially xylosylated glucan chain produced by XXT1 and/or XXT2. This suggests that XXT1 and XXT2 xylosylate a growing glucan chain to produce the GXXG repeat, which is then utilized by XXT5 to produce the biologically observed XXXG repeat of native xyloglucan present in most of the plants. These results allowed us to propose a model of sequential xylosylation of glucan chain synthesized by glucan synthase and support the synthesis of xyloglucan via multiprotein complex localized in plant Golgi as proposed previously

Building novel bioaugmentation consortia for degradation of polycyclic aromatic hydrocarbons: from selection to metabolic and genetic characterization of adaptive evolved microbial strains

Tese de mestrado em Microbiologia Aplicada, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, em 2018Os hidrocarbonetos aromáticos policíclicos (HAP) são poluentes ambientais encontrados na água, no solo e na atmosfera. Estes compostos são de elevada importância devido ao perigo que representam em termos de saúde humana e em termos ambientais, existindo uma lista de 16 HAP considerados poluentes prioritários, apresentando propriedades tóxicas, mutagénicas e/ou carcinogénicas. Por um lado, podem ser gerados naturalmente através de fogos florestais, atividade vulcânica e derramamentos de petróleo. Por outro lado, podem ter origem em atividades antropogénicas durante a combustão incompleta de materiais orgânicos, como o óleo, o petróleo ou a madeira. O facto de serem insolúveis em água dificulta a sua remoção e consequentemente leva a que se tornem recalcitrantes no meio ambiente, persistindo por muitos anos. Várias tecnologias de remediação têm sido testadas com o objetivo de remover estes contaminantes ambientais. Muitas das abordagens têm uma natureza química, o que além de se traduzir em elevados custos económicos também está associado à geração de subprodutos também de natureza tóxica. Recentemente, a biorremediação têm sido alvo de atenção já que o uso de microrganismos para remoção de HAP tem sido vista como uma opção mais segura e com menos custos. Vários géneros de bactérias Gram positivas e Gram negativas, bem como fungos e algas capazes de utilizar estes compostos como fonte de carbono e energia têm sido isolados e caraterizados. Na natureza, são raras as vezes que estes microrganismos com capacidade de degradar poluentes se encontram individualmente. Geralmente, existem em consórcio, ou seja, associações naturais de vários microrganismos diferentes que interagem sinergicamente entre si, aumentando a eficiência de degradação. Esta cooperação resulta por exemplo em interações onde os produtos metabólicos de um microrganismo podem ser o substrato para outro microrganismo. Nas estações de tratamento de águas residuais, o objetivo é obter um efluente que possa retornar ao ciclo da água com o mínimo impacto no ambiente. Neste contexto ecológico, os microrganismos geralmente realizam metabolismo aeróbio, tendo o oxigénio como aceitador final de eletrões. Catabolicamente, as enzimas com atividade de dioxigenase têm um papel fundamental na ativação do HAP para degradação. Um aspeto do metabolismo aeróbio é a formação de intermediários metabólicos como o salicilato, o catecol, o gentisato e o ftalato que após reações enzimáticas originaram metabolitos que compõem o ciclo dos ácidos tricarboxílicos. Estas proteínas com atividade catalítica estão amplamente distribuídas por diferentes microrganismos, considerando-se mesmo a existência de diferentes famílias de enzimas associadas a determinados taxa microbianos. Além disso, verifica-se uma plasticidade metabólica no que diz respeito à degradação destes compostos podendo ocorrer também degradação em condições de anaerobiose, tendo compostos inorgânicos como o sulfato, o nitrato e o ferro como aceitadores de eletrões. No entanto, ainda existe muito por revelar sobre este metabolismo. O presente trabalho tem como objetivo a seleção e caracterização metabólica e genética das melhores estirpes degradadoras presentes na coleção Biotask Bioremediation Culture, com vista à construção de um consórcio para degradação de HAPs, nomeadamente naftaleno, antraceno e fenantreno. Quarenta e sete estirpes microbianas provenientes de quatro experiências de evolução adaptativa e de vários ciclos dessas mesmas experiências tendo antraceno, fenantreno, óleo mineral e tristearina como única fonte de carbono e energia foram estudados. O inóculo inicial das experiências de evolução adaptativa foi proveniente de uma estação de tratamento de águas residuais. Após sete dias de crescimento das quarenta e sete estirpes tendo naftaleno, antraceno e fenantreno como única fonte de carbono e energia, foi realizada uma triagem das quarenta e sete estirpes através de uma avaliação direta e uma avaliação indireta da eficiência de degradação. Diretamente, foi desenvolvido e otimizado um método de quantificação por cromatografia líquida de elevada performance. Através da construção das curvas de calibração para naftaleno, antraceno e fenantreno foi possível dosear a quantidade desses poluentes presentes em amostras biológicas provenientes do crescimento. Indiretamente, foram realizados ensaios que permitiram concluir sobre a viabilidade celular através do método dos números mais prováveis dos microrganismos em estudo após os sete dias de crescimento. Desta triagem inicial, foi possível concluir que os melhores resultados foram obtidos para as estirpes provenientes do ciclo de evolução adaptativa mais recente, ou seja, foi corroborado o sucesso das experiências de evolução adaptativa. Surgiram também evidências da volatilidade e da toxicidade do naftaleno ser um fator que justifica a sua baixa concentração quantificada cromatograficamente e a baixa viabilidade dos microrganismos na presença deste HAP. Com vista a selecionar as estirpes com maior potencial degradativo foi realizada uma análise multivariada que integra os dados quantitativos da cromatografia líquida de elevada performance e os dados de viabilidade celular resultantes dos números mais prováveis. A análise em componentes principais permitiu escolher as estirpes que melhor se posicionavam no espaço formado pelas novas variáveis – componentes principais. Também esta análise permitiu observar que as estirpes pertencentes aos ciclos de evolução adaptativa mais recentes e que se esperam exibirem um melhor comportamento degradativo estavam localizadas na zona do gráfico onde seria expectável encontrar os melhores degradadores. Além das três estirpes selecionadas pela análise de componentes principais, num ensaio de crescimento surgiu um novo microrganismo como contaminante ambiental que foi adicionado à coleção já que exibia um comportamento degradativo em relação aos HAPs e dada a dificuldade em encontrar ao acaso microrganismos com essas caraterísticas (geralmente são isolados em locais já contaminados pelos poluentes), pensou-se ser interessante explorá-lo melhor e incluí-lo nos estudos futuros. Em seguida, os quatro microrganismos selecionados foram identificados presuntivamente através de sequenciação do gene bacteriano que codifica para o RNA ribossomal 16S como pertencentes aos géneros Pseudomonas (dois microrganismos), Acinetobacter e Paraburkholderia. Com o propósito de investigar o metabolismo degradativo dos HAPs por parte destas bactérias, foram realizados estudos de caraterização metabólica e genética. Em primeiro lugar, foi monitorizado durante quinze dias o crescimento destes microrganismos tendo naftaleno, antraceno ou fenantreno como única fonte de carbono e energia. As estirpes foram estudadas isoladamente e em conjunto, constituindo um consórcio bacteriano. Observou-se aparentemente um crescimento bifásico e a estirpe pertencente ao género Pseudomonas destacou-se, apresentando o melhor comportamento degradativo. O consórcio também revelou que as estirpes em conjunto apresentam resultados superiores do que se estiverem a crescer individualmente. Em segundo lugar, foi avaliada a produção de biosurfactantes como estratégia de solubilização destes compostos poluentes. Após uma pré-seleção com um teste qualitativo onde se observou a formação de uma emulsão estável em duas estirpes, foi realizado um ensaio quantitativo através da medição da tensão superficial. Foi possível concluir que a estirpe pertence ao género Acinetobacter aparentemente sintetiza algum composto com atividade surfactante. Em terceiro lugar, foi realizada uma pesquisa de genes que estão reportados como associados ao catabolismo dos PAHs através de polimerase chain reaction (PCR), tendo como alvo enzimas chave para o clivagem de intermediários metabólicos. Além disso, foi também monitorizado o crescimento tendo ftalato como única fonte de carbono e energia. Foi encontrada evidência que a estirpe pertencente ao género Acinetobacter possui genes que lhe permitem optar por um metabolismo aeróbio com clivagem orto do metabolito intermediário catecol. Quanto às estirpes pertences ao género Pseudomonas e ao género Paraburkholderia foi observada a presença de genes que lhe permitem seguir a via de degradação meta do catecol e ainda a via do gentisato. Em adição, a estirpe pertencente ao género Paraburkolderia revelou ser a única que claramente é capaz de crescer tendo ftalato como única fonte de carbono, o que indica que pode também seguir esta via degradativa. Relativamente ao outro microrganismo pertencente também ao género Pseudomonas não foi detetada nenhuma amplificação nem crescimento em ftalato. Em suma, pode concluir-se que o objetivo do trabalho foi atingido, tendo sido selecionadas e estudadas em termos metabólicos e genéticos as estirpes mais promissoras em termos de potencial de degradação de HAPs. Foi verificado o sucesso do crescimento das estirpes no consórcio e relevadas algumas características que podem explicar o seu comportamento degradativo. Mais estudos serão necessários para elucidar mais claramente quais as vias metabólicas do catabolismo destes compostos e consequentemente ter acesso ao máximo do seu potencial de degradação, com vista à construção de um consórcio eficaz para a biorremediação de locais contaminados.Polycyclic aromatic hydrocarbons (PAHs) are recalcitrant compounds considered as priority pollutants in soil, water and the atmosphere. They can have natural origin (fires, volcanic eruptions, among others) or anthropogenic origin by the incomplete combustion of organic matter (coal, oil, wood). This is not only an environmental problem but also impacts on public health, since studies that indicate the toxicity and mutagenic/carcinogenic effects of PAHs are reported. Many attempts have been made to develop strategies to eliminate these compounds. Bioremediation has emerged as a possible solution to the problem using microorganisms and taking advantage of their degrading capacity to reduce or eliminate the presence of PAHs. The presence of PAHs on planet Earth through the evolution of life shaped the evolution of metabolic pathways allowing microorganisms to use these compounds as carbon and energy sources. A screening of forty-seven strains (Biotask Bioremediation Culture collection) from adaptive evolution experiments was performed. The initial inoculum of the experiments was from a wastewater treatment plant. In order to evaluate the degradative potential of these strains, growth assays were performed with naphthalene, anthracene and phenanthrene as the only source of carbon and energy. Directly, an HPLC method was developed and optimized that allowed the quantification of PAHs present in the biological samples. Indirectly, a cell viability study was performed through the most probable numbers. The most recent cycle of adaptive evolution revealed the best results as expected. The selection of the best degraders was performed through a principal component analysis. In addition, an environmental contaminant revealed an interesting degradative behaviour and was included in the BBC collection. Sequencing of the bacterial 16S rRNA gene, allowed the four strains to be presumptively identified as belonging to the genus Pseudomonas, Acinetobacter and Paraburkholderia. Characterization of the strains involved three phases. Initially, the growth of the microorganisms was monitored having the PAHs as the carbon source for 15 days, both individually and in a consortium. The strain belonging to the genus Pseudomonas present the best results and possibly a bi-phasic growth. The consortium also revealed that strains together yield better results than individual growth. Then, the production of biosurfactants was evaluated qualitatively through the observation of an emulsion and quantitatively through the measurement of surface tension. The strain belonging to the genus Acinetobacter was the only one that apparently synthesizes a compound with surfactant activity. Finally, the presence of genes associated with PAH catabolism and growth having the metabolic intermediate phthalate as the sole source of carbon was evaluated. The strains belonging to the genera Pseudomonas and Paraburkholderia were the ones that presented greater metabolic plasticity. In summary, it can be concluded that the objective of the work was achieved, and the most promising strains with higher potential for degradation of PAHs were selected and studied in metabolic and genetic terms. The growth of the strains in the consortium was successfully achieved and some characteristics that may explain strains degradative behaviour were revealed. More studies will be needed to elucidate more clearly the whole process in order to build an effective consortium for the bioremediation of contaminated sites

Structural and Functional Analysis of Grapefruit Flavonol-Specific-3-O-GT Mutant P145T

This research is focused on the study of the effect of mutating proline 145 to threonine on the substrate and regiospecificity of flavonol specific 3-O-glucosyltransferase (Cp3GT). While the mutant P145T enzyme did not glucosylate anthocyanidins, it did glucosylate flavanones and flavones in addition to retaining activity with flavonols. HPLC was used for product identification and showed mutant P145T glucosylated naringenin at the 7-OH position forming naringenin-7-O-glucoside and flavonols at the 3-OH position. Homology modeling and docking was done to predict the acceptor substrate recognition pattern and models were validated by experimental results. In other related work, a thrombin cleavage site was inserted into wild type Cp3GT and recombinant P145T enzyme between the enzyme and the C-myc tags in order to be able to cleave off tags. This provides the tool needed for future efforts to crystallize these proteins for structural determination

Flexible Modellerweiterung und Optimierung von Erdbebensimulationen

Simulations of realistic earthquake scenarios require scalable software and extensive supercomputing resources. With increasing fidelity in simulations, advanced rheological and source models need to be incorporated. I introduce a domain-specific language in order to handle the model flexibility in combination with the high efficiency requirements. The contributions in this thesis enabled the to date largest and longest dynamic rupture simulation of the 2004 Sumatra earthquake.Realistische Erdbebensimulationen benötigen skalierbare Software und beträchtliche Rechenressourcen. Mit zunehmender Genauigkeit der Simulationen müssen fortschrittliche rheologische und Quellmodelle integriert werden. Ich führe eine domänenspezifische Sprache ein, um die Modelflexibilität in Kombination mit den hohen Effizienzanforderungen zu beherrschen. Die Beiträge in dieser Arbeit haben die bisher größte und längste dynamische Bruchsimulation des Sumatra-Erdbebens von 2004 ermöglicht

NKS1/ELMO4 is an integral protein of a pectin synthesis protein complex and maintains Golgi morphology and cell adhesion in Arabidopsis

Adjacent plant cells are connected by specialized cell wall regions, called middle lamellae, which influence critical agricultural characteristics, including fruit ripening and organ abscission. Middle lamellae are enriched in pectin polysaccharides, specifically homogalacturonan (HG). Here, we identify a plant- specific Arabidopsis DUF1068 protein, called NKS1/ELMO4, that is required for middle lamellae integrity and cell adhesion. NKS1 localizes to the Golgi apparatus and loss of NKS1 results in changes to Golgi structure and function. The nks1 mutants also display HG deficient phenotypes, including reduced seedling growth, changes to cell wall composition, and tissue integrity defects. These phenotypes are comparable to qua1 and qua2 mutants, which are defective in HG biosynthesis. Notably, genetic interactions indicate that NKS1 and the QUAs work in a common pathway. Protein interaction analyses and modeling corroborate that they work together in a stable protein complex with other pectin- related proteins. We propose that NKS1 is an integral part of a large pectin synthesis protein complex and that proper function of this complex is important to support Golgi structure and function. Significance Cell walls are essential to cell morphogenesis, to protect plants against environmental stress, and for an array of products in our daily life. Understanding how plants produce cell wall polymers is therefore important. In this study, we outline how a family of unknown proteins function as a scaffold for key synthesis components of pectin, a central cell wall polymer. Our results thus define a robust pectin synthesis protein complex that is essential for the structure and function of Golgi and for plant tissue integrity. These results add critical information regarding pectin synthesis and cell wall metabolons

Efficient integration of software components for scientific simulations

Abstract unavailable please refer to PD

Study and characterization of Glycosyltransferases from Paramecium bursaria Chlorella virus – 1

openGiant Viruses are a class of uncommon cellular parasites discovered about 30 years ago1. They are defined as Nucleo Cytoplasmic Large DNA viruses (NCLDVs) according to the notable viral particle dimensions (about 400nm) and the genome complexity. In addition, NCLDVs possess genes with “cell-like properties”, that allow the virus to be, at least in part, independent from the host molecular mechanisms1. One of the most important pathways that is almost totally encoded by NCLDVs is the glycosylation. Generally, viruses use the ER/Golgi compartments of the host to glycosylate their own proteins. For NCLDVs, an almost complete system to elongate, modify and synthetize the glycoforms is set up in the viral factories, which are defined structures in the host cytoplasm. The topic of this work was the study and the characterization of two of the six putative glycosyltransferases (GTs) from Paramecium bursaria Chlorella virus- 1 (PBCV-1): A064R and A075L2. PBCV-1 possesses in fact an highly glycosylated capsid that displays uncommon glycoforms only shared by chloroviruses3. The identification of the glycoform structure suggest that they are probably synthetized by the virus and not by the host. These findings represented the starting point to analyse PBCV-1 genome looking for genes encoding those enzymes. In the present work, A064R is characterized by enzymatic analysis, demonstrating that it is a multidomain enzyme, with two rhamnosyltransferase activities and a methyltransferase one. A075L is also demonstrated to be a GT, by enzymatic analysis and ITC experiments. Experiments aimed also to identify the 3D structure of the protein, and to confirm its interaction with the substrate, the UDP-xylose. The solving of the 3D structure and the enzymatic characterisation are currently underway. A064R and A075L enzymes display interesting catalytic properties that could be explored for biotechnological applications. In fact, the study of the enzymes that process glycans is a recent topic explored for the production of compounds largely used as bioactive molecules 4. Identification of novel GTs will provide new tools that can expand the biological biodiversity of glycans as bioactive natural products, which is well known to participate in the molecules drug efficiency in terms of pharmacokinetics and pharmacodynamics4, and that could be also exploited for the production of new carbohydrate vaccines.  embargoed_20210130XXXII CICLO - MEDICINA SPERIMENTALE - BiochimicaLaugieri, MARIA ELEN

A Compass to Controlled Graph Rewriting

With the growing complexity and autonomy of software-intensive systems, abstract modeling to study and formally analyze those systems is gaining on importance. Graph rewriting is an established, theoretically founded formalism for the graphical modeling of structure and behavior of complex systems. A graph-rewriting system consists of declarative rules, providing templates for potential changes in the modeled graph structures over time. Nowadays complex software systems, often involving distributedness and, thus, concurrency and reactive behavior, pose a challenge to the hidden assumption of global knowledge behind graph-based modeling; in particular, describing their dynamics by rewriting rules often involves a need for additional control to reflect algorithmic system aspects. To that end, controlled graph rewriting has been proposed, where an external control language guides the sequence in which rules are applied. However, approaches elaborating on this idea so far either have a practical, implementational focus without elaborating on formal foundations, or a pure input-output semantics without further considering concurrent and reactive notions. In the present thesis, we propose a comprehensive theory for an operational semantics of controlled graph rewriting, based on well-established notions from the theory of process calculi. In the first part, we illustrate the aforementioned fundamental phenomena by means of a simplified model of wireless sensor networks (WSN). After recapitulating the necessary background on DPO graph rewriting, the formal framework used throughout the thesis, we present an extensive survey on the state of the art in controlled graph rewriting, along the challenges which we address in the second part where we elaborate our theoretical contributions. As a novel approach, we propose a process calculus for controlled graph rewriting, called RePro, where DPO rule applications are controlled by process terms closely resembling the process calculus CCS. In particular, we address the aforementioned challenges: (i) we propose a formally founded control language for graph rewriting with an operational semantics, (ii) explicitly addressing concurrency and reactive behavior in system modeling, (iii) allowing for a proper handling of process equivalence and action independence using process-algebraic notions. Finally, we present a novel abstract verification approach for graph rewriting based on abstract interpretation of reactive systems. To that end, we propose the so-called compasses as an abstract representation of infinite graph languages and demonstrate their use for the verification of process properties over infinite input sets