150 research outputs found

    Characterization of new parts for synthetic biology applications in bacteria and yeast supported by automated methods

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    The recently presented IPCC report once again urges us to intensify efforts to build a sustainable economy. The UN also emphasizes that most of the economy still operates in a linear model and urgently needs to be reformed. Biotechnological applications can offer a valuable contribution, as they can replace energyintensive processes in the chemical industry. In addition, they have the charm that the growth of the used organisms is not dependent on crude oil sources. Further, they can use inexpensive energy sources from industrial by-products as energy sources. The oil yeast Yarrowia lipolytica in particular is emerging as a valuable workhorse for industry and science. Due to the natural accumulation of lipids, the yeast represents an interesting production host for industrial applications. To date, unfortunately, only a limited repertoire of metabolic engineering approaches or SynBio circuits is available for yeast. This thesis aims, among other things, to expand the portfolio of SynBio parts for this yeast. To this end, chapter 3 presents ways to further exploit the organism. Thus, a broad-hostrange plasmid (section 3.3.1) was developed, which allows rapid testing of constructs in three organisms. This plasmid could be used to test a tetracycline aptamer in Y. lipolytica (section 3.3.2). The aptamer was previously developed for Saccharomyces cerevisiae and could be successfully used for translation inactivation. Y. lipolytica was successfully modified with a monomer of the aptamer. Unfortunately, growth assays revealedthat the strain carrying the aptamer was severely impaired in fitness. Thus, a clear conclusion about the functionality was not possible. An expansion of the repertoire of Y. lipolytica and other organisms was pursued by establishing artificial landing pads (section 3.3.3). The computer-generated DNA sequences were designed to cause as little offtarget effects as possible when using Cas9 in the organisms Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica. To this end, up to 25 artificial protospacers were lined up in a sequence up to 900 bp long. These were successfully integrated in the genome of the organisms. Experiments with CRISPRi and CRISPRa served as proof-of-concept. By addressing different protospacers, different expression levels should be achieved depending on the distance between protospacer and reporter gene. Fine-graded activation of expression was demonstrated in S. cerevisiae. The use of CRISPRa increased the basal activity of the minimal promoter by 3.4-fold. In Y. lipolytica, there was no clear gradient as in S. cerevisiae. The effect of CRISPRi in B. subtilis was dependent on the promoter used. Automation methods were developed to support the molecular biology and microbiology work (chapter 4). Due to a clearly visible edge effect when using the incubator of the CompuGene Robotics platform, a method was developed to randomize the samples in the microtiter plates (section 4.4). Here it was shown that this method cannot eliminate the edge effect, but it can improve comparability between samples. In addition, a method was developed to use the liquid handler of the automation platform to pick colonies from agar plates (section 4.5)

    Position Paper on Dataset Engineering to Accelerate Science

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    Data is a critical element in any discovery process. In the last decades, we observed exponential growth in the volume of available data and the technology to manipulate it. However, data is only practical when one can structure it for a well-defined task. For instance, we need a corpus of text broken into sentences to train a natural language machine-learning model. In this work, we will use the token \textit{dataset} to designate a structured set of data built to perform a well-defined task. Moreover, the dataset will be used in most cases as a blueprint of an entity that at any moment can be stored as a table. Specifically, in science, each area has unique forms to organize, gather and handle its datasets. We believe that datasets must be a first-class entity in any knowledge-intensive process, and all workflows should have exceptional attention to datasets' lifecycle, from their gathering to uses and evolution. We advocate that science and engineering discovery processes are extreme instances of the need for such organization on datasets, claiming for new approaches and tooling. Furthermore, these requirements are more evident when the discovery workflow uses artificial intelligence methods to empower the subject-matter expert. In this work, we discuss an approach to bringing datasets as a critical entity in the discovery process in science. We illustrate some concepts using material discovery as a use case. We chose this domain because it leverages many significant problems that can be generalized to other science fields.Comment: Published at 2nd Annual AAAI Workshop on AI to Accelerate Science and Engineering (AI2ASE) https://ai-2-ase.github.io/papers/16%5cSubmission%5cAAAI_Dataset_Engineering-8.pd

    Holm oak decline is determined by shifts in fine root phenotypic plasticity in response to belowground stress

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    Climate change and pathogen outbreaks are the two major causes of decline in Mediterranean holm oak trees (Quercus ilex L. subsp. ballota (Desf.) Samp.). Crown-level changes in response to these stressful conditions have been widely documented but the responses of the root systems remain unexplored. The effects of environmental stress over roots and its potential role during the declining process need to be evaluated. We aimed to study how key morphological and architectural root parameters and nonstructural carbohydrates of roots are affected along a holm oak health gradient (i.e. within healthy, susceptible and declining trees). Holm oaks with different health statuses had different soil resource-uptake strategies. While healthy and susceptible trees showed a conservative resource-uptake strategy independently of soil nutrient availability, declining trees optimized soil resource acquisition by increasing the phenotypic plasticity of their fine root system. This increase in fine root phenotypic plasticity in declining holm oaks represents an energy-consuming strategy promoted to cope with the stress and at the expense of foliage maintenance. Our study describes a potential feedback loop resulting from strong unprecedented belowground stress that ultimately may lead to poor adaptation and tree death in the Spanish dehesa.This research was mainly funded by the Spanish Government through the IBERYCA project (CGL2017-84723-P), its associated FPI scholarship BES-2014-067971 (ME-V) and SMARTSOIL (PID2020-113244GB-C21). It was further supported by the BC3 María de Maeztu excellence accreditation (MDM-2017-0714; the Spanish Government) and by the BERC 2018–2021 and the UPV/EHU-GV IT-1018-16 programme (Basque Government). Additionally, this research was further supported through the ‘Juan de la Cierva programme’ (MV; IJCI-2017-34640; the Spanish Government) and one project funded by the Romanian Ministry of Research, Innovation and Digitization through UEFISCDI (A-MH; REASONING, PN-III-P1-1.1-TE-2019-1099)

    Hybrid modelling of biological systems: current progress and future prospects

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    Copyright © 2022 The Author(s). Integrated modelling of biological systems is becoming a necessity for constructing models containing the major biochemical processes of such systems in order to obtain a holistic understanding of their dynamics and to elucidate emergent behaviours. Hybrid modelling methods are crucial to achieve integrated modelling of biological systems. This paper reviews currently popular hybrid modelling methods, developed for systems biology, mainly revealing why they are proposed, how they are formed from single modelling formalisms and how to simulate them. By doing this, we identify future research requirements regarding hybrid approaches for further promoting integrated modelling of biological systems.National Natural Science Foundation of China (61873094)

    Virginia Woolf’s futures, 1934-41: substrate, archive, anterior

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    My thesis discusses Virginia Woolf’s late manuscript and typescript drafts. I locate the start of Woolf’s ‘late’ work in January 1934, as she drafts the 1917 chapter of The Years. My thesis argues that this draft signals a ‘reparative’ turn characterized by a change in her thinking of the future. I use the term ‘reparative’ after Eve Kosofsky Sedgwick’s anatomy of paranoid and reparative modes of thought. I use these late draft works to make the case that, from January 1934, Woolf starts to imagine new futures premised on genuine difference, futures that offer an alternative to patriarchy, to fascism, and to war, and I further argue that Woolf uses the textual space afforded her by the draft page as a locus to imagine such futures. My reading of Woolf’s draft material is informed by the words ‘substrate,’ ‘archive’ and ‘anterior’. Substrate is a term from Jacques Derrida’s Archive Fever: A Freudian Impression (1995), a work that I use as a key analytic for my work in Woolf’s archive. The term ‘substrate’ signifies a surface for inscription: any surface, any inscription. I use this term to engage with the material specificity of Woolf’s draft pages and with the material specificity of my own work in the Woolfian archive. ‘Archive’ is the second word that guides this thesis: on the one hand it is the simplest, but I work throughout this thesis to problematise the notion of the archive as a fixed repository for documents, using Archive Fever both as a guide through Woolf’s archive and as a way to radically expand its parameters. ‘Anterior’ is a Janus-faced tense structure that speaks at once to what will have happened and to a past that precedes what has happened: it is my contention that this tense structure is apt to the archival work of genetic critics and that Woolf’s writing on the future gestures towards anteriority. This thesis is divided into three parts. Part One is introductory, providing a methodological and theoretical justification for the archival work I present later in the thesis. In Chapter One, I provide a close reading of the opening of ‘Time Passes,’ the second part of To the Lighthouse (1927), in order to establish key tropes in my thesis. I read both the published version(s) of this text and its avant-textes, as well as exogenetic material including Virgil’s Georgics, which I argue is an intertext to ‘Time Passes,’ in order to introduce key theoretical and methodological tenets of my thesis. Chapter Two expands this reading to encompass Woolf’s use of the word ‘future’ prior to 1934, tracing its changing valences by means of distant reading. This distant reading provides the groundwork for my theorising of a reparative turn in the early months of 1934. Part Two reads the holograph drafts of the ‘1917’ chapter of The Years as a richly generative avant-texte not only for Woolf’s 1937 novel but also for Three Guineas (1938). Chapter Three examines tropes and figurations common to both the draft scene and to Three Guineas, including an early and textually fraught invocation of Three Guineas’ Society of Outsiders and a call to revolutionary arson at Oxford and Cambridge. I read between the 1934 draft and the published texts of 1937 and 1938, alongside Derrida’s Archive Fever in order to expand the theoretical boundaries of Woolf’s archive. Chapter Four offers a genetic reading of Antigone’s footfalls from the 1934 draft through the published texts of 1937 and 1938 and reads Woolf’s interaction with Sophocles’ drama in all three texts. I light on a curious instantiation of Antigone’s speech—a misquotation of the ‘five words’ upon which Three Guineas tropes—in the draft, reading it alongside the children’s chorus at the end of The Years. Part Three of my thesis constellates the draft fragments of ‘Anon’ and ‘The Reader’ with Between the Acts (1941). Chapter Five examines the fragments’ curatorial and editorial history, using a startling collocation of a single page from this part of Woolf’s archive in order to argue that closer critical attention needs to be paid to the material form of the fragments and the archival, curatorial, and editorial context in which contemporary readers encounter them. Chapter Six investigates modes of anonymity in Between the Acts and ‘Anon’ and ‘The Reader.’ I use three definitions of the word ‘anon,’ the first an abbreviation for ‘anonymous,’ the second an obsolete word meaning ‘of one body,’ and the third meaning ‘now again’ to read Woolf’s final constellation of works as theorising anonymity as a mode of subjectivity that is profoundly future-oriented

    How to Achieve Inclusive Growth

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    Rising inequality and widespread poverty, social unrest and polarization, gender and ethnic disparities, declining social mobility, economic fragility, unbalanced growth due to technology and globalization, and existential danger from climate change are urgent global concerns of our day. These issues are intertwined. They therefore require a holistic framework to examine their interplay and bring the various strands together. This book brings together leading academic economists and experts from several international institutions to explain the sources and scale of these challenges. The book summarizes a wide array of empirical evidence and country experiences, lays out practical policy solutions, and devises a comprehensive and unified plan of action for combatting these economic and social disparities. This authoritative book is accessible to policy makers, students, and the general public interested in how to craft a brighter future by building a sustainable, green, and inclusive society in the years ahead

    Faktor Pengaruh Bahasa Inggeris dalam Pembelajaran Bahasa Mandarin Sebagai Bahasa Asing Nur Farrah Atiqah Amran & Muhammad Nazirul Mohd Noor

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    Salah satu nilai tambah yang amat diperlukan pada masa kini ialah penguasaan bahasa asing. Pelan Pembangunan Pendidikan Malaysia 2013-2015 telah mengetengahkan pembelajaran bahasa ketiga. Menurut Teori Bahasa Halliday, kanak-kanak menjadi mahir dalam bahasa kerana mereka perlu berinteraksi dengan orang di persekitaran mereka. Ini bermakna untuk memenuhi keperluan asas mereka, kanak-kanak itu perlu berinteraksi dengan orang ramai pada peringkat awal. Fenomena ini tidak berlaku dalam sampel. Oleh itu, pelajar Melayu yang hanya berbahasa Melayu dan Inggeris tidak akan mempelajari bahasa Mandarin dengan baik. Kajian awal ini bertujuan untuk menganalisis faktor-faktor yang menyumbang kepada pengaruh bahasa Inggeris dalam kalangan pelajar Melayu semasa mempelajari bahasa Mandarin. Sampel kajian melibatkan 10 orang pelajar Melayu di sebuah universiti awam. Instrumen yang digunakan dalam kajian ini ialah soal selidik. Dapatan kajian ini diharapkan dapat membantu ahli akademik dalam melahirkan graduan yang berkualiti tinggi dengan kebolehan bertutur dalam bahasa ketiga dengan fasih. Selain itu, ia juga akan meningkatkan reputasi universiti dengan pencapaian pelajar yang cemerlang, sekali gus memberi manfaat kepada pembangunan negara khususnya dalam hubungan antarabangsa

    An Imaging Mass Spectrometry Investigation Into the N-linked Glycosylation Landscape of Pancreatic Ductal Adenocarcinoma and the Development of Associated Tools for Enhanced Glycan Separation and Characterization

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    The severity of pancreatic ductal adenocarcinoma (PDAC) is largely attributed to a failure to detect the disease before metastatic spread has occurred. CA19-9, a carbohydrate biomarker, is used clinically to surveille disease progression, but due to specificity challenges is not suitable for early discovery. As CA19-9 and other prospective markers are glycan epitopes, there is great clinical interest in understanding the glycobiology of pancreatic cancer. Unfortunately, few studies have been able to link glycosylation changes directly to pancreatic tumors and instead have focused on peripheral glycan alterations in the serum of PDAC patients. To address this gap in our understanding, we applied an imaging mass spectrometry (IMS) approach with complementary enzymatic and chemical isomer separation techniques to spatially assess the PDAC N-glycome in a cohort of pancreatic cancer patients. Orthogonally, we characterized the expression of CA19-9 and a new biomarker, sTRA, by multi-round immunofluorescence (IF) in the same cohort. These analyses revealed increased sialylation, fucosylation and branching amongst other structural themes in areas of PDAC tumor tissue. CA19-9 expressing tumors were defined by multiply branched, fucosylated bisecting N-glycans while sTRA expressing tumors favored tetraantennary N-glycans with polylactosamine extensions. IMS and IF-derived glycan and biomarker features were used to build classification models that detected PDAC tissue with an AUC of 0.939, outperforming models using either dataset individually. While studying sialylation isomers in our PDAC cohort, we saw an opportunity to enhance the chemical derivatization protocol we were using to address its shortcomings and expand its functionality. Subsequently, we developed a set of novel amidation-amidation strategies to stabilize and differentially label 2,3 and 2,6-linked sialic acids. In our alkyne-based approach, the differential mass shifts induced by the reactions allow for isomeric discrimination in imaging mass spectrometry experiments. This scheme, termed AAXL, was further characterized in clinical tissue specimens, biofluids and cultured cells. Our azide-based approach, termed AAN3, was more suitable for bioorthogonal applications, where the azide tag installed on 2,3 and 2,8-sialic acids could be reacted by click chemistry with a biotin-alkyne for subsequent streptavidin-peroxidase staining. Furthering the use of AAN3, we developed two additional techniques to fluorescently label (SAFER) and preferentially enrich (SABER) 2,3 and 2,8-linked sialic acids for more advanced glycomic applications. Initial experiments with these novel approaches have shown successful fluorescent staining and the identification of over 100 sialylated glycoproteins by LC-MS/MS. These four bioorthogonal strategies provide a new glycomic tool set for the characterization of sialic acid isomers in pancreatic and other cancers. Overall, this work furthers our collective understanding of the glycobiology underpinning pancreatic cancer and potentiates the discovery of novel carbohydrate biomarkers for the early detection of PDAC

    Advances in Condition Monitoring, Optimization and Control for Complex Industrial Processes

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    The book documents 25 papers collected from the Special Issue “Advances in Condition Monitoring, Optimization and Control for Complex Industrial Processes”, highlighting recent research trends in complex industrial processes. The book aims to stimulate the research field and be of benefit to readers from both academic institutes and industrial sectors
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