1,887 research outputs found

    Converging organoids and extracellular matrix::New insights into liver cancer biology

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    Converging organoids and extracellular matrix::New insights into liver cancer biology

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    Primary liver cancer, consisting primarily of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), is a heterogeneous malignancy with a dismal prognosis, resulting in the third leading cause of cancer mortality worldwide [1, 2]. It is characterized by unique histological features, late-stage diagnosis, a highly variable mutational landscape, and high levels of heterogeneity in biology and etiology [3-5]. Treatment options are limited, with surgical intervention the main curative option, although not available for the majority of patients which are diagnosed in an advanced stage. Major contributing factors to the complexity and limited treatment options are the interactions between primary tumor cells, non-neoplastic stromal and immune cells, and the extracellular matrix (ECM). ECM dysregulation plays a prominent role in multiple facets of liver cancer, including initiation and progression [6, 7]. HCC often develops in already damaged environments containing large areas of inflammation and fibrosis, while CCA is commonly characterized by significant desmoplasia, extensive formation of connective tissue surrounding the tumor [8, 9]. Thus, to gain a better understanding of liver cancer biology, sophisticated in vitro tumor models need to incorporate comprehensively the various aspects that together dictate liver cancer progression. Therefore, the aim of this thesis is to create in vitro liver cancer models through organoid technology approaches, allowing for novel insights into liver cancer biology and, in turn, providing potential avenues for therapeutic testing. To model primary epithelial liver cancer cells, organoid technology is employed in part I. To study and characterize the role of ECM in liver cancer, decellularization of tumor tissue, adjacent liver tissue, and distant metastatic organs (i.e. lung and lymph node) is described, characterized, and combined with organoid technology to create improved tissue engineered models for liver cancer in part II of this thesis. Chapter 1 provides a brief introduction into the concepts of liver cancer, cellular heterogeneity, decellularization and organoid technology. It also explains the rationale behind the work presented in this thesis. In-depth analysis of organoid technology and contrasting it to different in vitro cell culture systems employed for liver cancer modeling is done in chapter 2. Reliable establishment of liver cancer organoids is crucial for advancing translational applications of organoids, such as personalized medicine. Therefore, as described in chapter 3, a multi-center analysis was performed on establishment of liver cancer organoids. This revealed a global establishment efficiency rate of 28.2% (19.3% for hepatocellular carcinoma organoids (HCCO) and 36% for cholangiocarcinoma organoids (CCAO)). Additionally, potential solutions and future perspectives for increasing establishment are provided. Liver cancer organoids consist of solely primary epithelial tumor cells. To engineer an in vitro tumor model with the possibility of immunotherapy testing, CCAO were combined with immune cells in chapter 4. Co-culture of CCAO with peripheral blood mononuclear cells and/or allogenic T cells revealed an effective anti-tumor immune response, with distinct interpatient heterogeneity. These cytotoxic effects were mediated by cell-cell contact and release of soluble factors, albeit indirect killing through soluble factors was only observed in one organoid line. Thus, this model provided a first step towards developing immunotherapy for CCA on an individual patient level. Personalized medicine success is dependent on an organoids ability to recapitulate patient tissue faithfully. Therefore, in chapter 5 a novel organoid system was created in which branching morphogenesis was induced in cholangiocyte and CCA organoids. Branching cholangiocyte organoids self-organized into tubular structures, with high similarity to primary cholangiocytes, based on single-cell sequencing and functionality. Similarly, branching CCAO obtain a different morphology in vitro more similar to primary tumors. Moreover, these branching CCAO have a higher correlation to the transcriptomic profile of patient-paired tumor tissue and an increased drug resistance to gemcitabine and cisplatin, the standard chemotherapy regimen for CCA patients in the clinic. As discussed, CCAO represent the epithelial compartment of CCA. Proliferation, invasion, and metastasis of epithelial tumor cells is highly influenced by the interaction with their cellular and extracellular environment. The remodeling of various properties of the extracellular matrix (ECM), including stiffness, composition, alignment, and integrity, influences tumor progression. In chapter 6 the alterations of the ECM in solid tumors and the translational impact of our increased understanding of these alterations is discussed. The success of ECM-related cancer therapy development requires an intimate understanding of the malignancy-induced changes to the ECM. This principle was applied to liver cancer in chapter 7, whereby through a integrative molecular and mechanical approach the dysregulation of liver cancer ECM was characterized. An optimized agitation-based decellularization protocol was established for primary liver cancer (HCC and CCA) and paired adjacent tissue (HCC-ADJ and CCA-ADJ). Novel malignancy-related ECM protein signatures were found, which were previously overlooked in liver cancer transcriptomic data. Additionally, the mechanical characteristics were probed, which revealed divergent macro- and micro-scale mechanical properties and a higher alignment of collagen in CCA. This study provided a better understanding of ECM alterations during liver cancer as well as a potential scaffold for culture of organoids. This was applied to CCA in chapter 8 by combining decellularized CCA tumor ECM and tumor-free liver ECM with CCAO to study cell-matrix interactions. Culture of CCAO in tumor ECM resulted in a transcriptome closely resembling in vivo patient tumor tissue, and was accompanied by an increase in chemo resistance. In tumor-free liver ECM, devoid of desmoplasia, CCAO initiated a desmoplastic reaction through increased collagen production. If desmoplasia was already present, distinct ECM proteins were produced by the organoids. These were tumor-related proteins associated with poor patient survival. To extend this method of studying cell-matrix interactions to a metastatic setting, lung and lymph node tissue was decellularized and recellularized with CCAO in chapter 9, as these are common locations of metastasis in CCA. Decellularization resulted in removal of cells while preserving ECM structure and protein composition, linked to tissue-specific functioning hallmarks. Recellularization revealed that lung and lymph node ECM induced different gene expression profiles in the organoids, related to cancer stem cell phenotype, cell-ECM integrin binding, and epithelial-to-mesenchymal transition. Furthermore, the metabolic activity of CCAO in lung and lymph node was significantly influenced by the metastatic location, the original characteristics of the patient tumor, and the donor of the target organ. The previously described in vitro tumor models utilized decellularized scaffolds with native structure. Decellularized ECM can also be used for creation of tissue-specific hydrogels through digestion and gelation procedures. These hydrogels were created from both porcine and human livers in chapter 10. The liver ECM-based hydrogels were used to initiate and culture healthy cholangiocyte organoids, which maintained cholangiocyte marker expression, thus providing an alternative for initiation of organoids in BME. Building upon this, in chapter 11 human liver ECM-based extracts were used in combination with a one-step microfluidic encapsulation method to produce size standardized CCAO. The established system can facilitate the reduction of size variability conventionally seen in organoid culture by providing uniform scaffolding. Encapsulated CCAO retained their stem cell phenotype and were amendable to drug screening, showing the feasibility of scalable production of CCAO for throughput drug screening approaches. Lastly, Chapter 12 provides a global discussion and future outlook on tumor tissue engineering strategies for liver cancer, using organoid technology and decellularization. Combining multiple aspects of liver cancer, both cellular and extracellular, with tissue engineering strategies provides advanced tumor models that can delineate fundamental mechanistic insights as well as provide a platform for drug screening approaches.<br/

    Natural and Technological Hazards in Urban Areas

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    Natural hazard events and technological accidents are separate causes of environmental impacts. Natural hazards are physical phenomena active in geological times, whereas technological hazards result from actions or facilities created by humans. In our time, combined natural and man-made hazards have been induced. Overpopulation and urban development in areas prone to natural hazards increase the impact of natural disasters worldwide. Additionally, urban areas are frequently characterized by intense industrial activity and rapid, poorly planned growth that threatens the environment and degrades the quality of life. Therefore, proper urban planning is crucial to minimize fatalities and reduce the environmental and economic impacts that accompany both natural and technological hazardous events

    LIPIcs, Volume 251, ITCS 2023, Complete Volume

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    LIPIcs, Volume 251, ITCS 2023, Complete Volum

    Improving students' code correctness and test completeness by informal specifications

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    The quality of software produced by students is often poor. How to teach students to develop good quality software has long been a topic in computer science education and research. We must conclude that we still do not have a good answer to this question. Specifications are necessary to determine the correctness of software, to develop error-free software and to write complete tests. Several attempts have been made to teach students to write specifications before writing code. So far, that has not proven to be very successful: Students do not like to write a specification and do not see the benefits of writing specifications. In this paper we focus on the use of informal specifications. Instead of teaching students how to write specifications, we teach them how to use informal specifications to develop correct software. The results were surprising: the number of errors in software and the completeness of tests both improved considerably and, most importantly, students really appreciate the specifications. We think that if students appreciate specification, we have a key to teach them how to specify and to appreciate its value.Comment: 14 page

    ENGINEERING HIGH-RESOLUTION EXPERIMENTAL AND COMPUTATIONAL PIPELINES TO CHARACTERIZE HUMAN GASTROINTESTINAL TISSUES IN HEALTH AND DISEASE

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    In recent decades, new high-resolution technologies have transformed how scientists study complex cellular processes and the mechanisms responsible for maintaining homeostasis and the emergence and progression of gastrointestinal (GI) disease. These advances have paved the way for the use of primary human cells in experimental models which together can mimic specific aspects of the GI tract such as compartmentalized stem-cell zones, gradients of growth factors, and shear stress from fluid flow. The work presented in this dissertation has focused on integrating high-resolution bioinformatics with novel experimental models of the GI epithelium systems to describe the complexity of human pathophysiology of the human small intestines, colon, and stomach in homeostasis and disease. Here, I used three novel microphysiological systems and developed four computational pipelines to describe comprehensive gene expression patterns of the GI epithelium in various states of health and disease. First, I used single cell RNAseq (scRNAseq) to establish the transcriptomic landscape of the entire epithelium of the small intestine and colon from three human donors, describing cell-type specific gene expression patterns in high resolution. Second, I used single cell and bulk RNAseq to model intestinal absorption of fatty acids and show that fatty acid oxidation is a critical regulator of the flux of long- and medium-chain fatty acids across the epithelium. Third, I use bulk RNAseq and a machine learning model to describe how inflammatory cytokines can regulate proliferation of intestinal stem cells in an experimental model of inflammatory hypoxia. Finally, I developed a high throughput platform that can associate phenotype to gene expression in clonal organoids, providing unprecedented resolution into the relationship between comprehensive gene expression patterns and their accompanying phenotypic effects. Through these studies, I have demonstrated how the integration of computational and experimental approaches can measurably advance our understanding of human GI physiology.Doctor of Philosoph

    Ny forståelse av gasshydratfenomener og naturlige inhibitorer i råoljesystemer gjennom massespektrometri og maskinlæring

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    Gas hydrates represent one of the main flow assurance issues in the oil and gas industry as they can cause complete blockage of pipelines and process equipment, forcing shut downs. Previous studies have shown that some crude oils form hydrates that do not agglomerate or deposit, but remain as transportable dispersions. This is commonly believed to be due to naturally occurring components present in the crude oil, however, despite decades of research, their exact structures have not yet been determined. Some studies have suggested that these components are present in the acid fractions of the oils or are related to the asphaltene content of the oils. Crude oils are among the worlds most complex organic mixtures and can contain up to 100 000 different constituents, making them difficult to characterise using traditional mass spectrometers. The high mass accuracy of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) yields a resolution greater than traditional techniques, making FT-ICR MS able to characterise crude oils to a greater extent, and possibly identify hydrate active components. FT-ICR MS spectra usually contain tens of thousands of peaks, and data treatment methods able to find underlying relationships in big data sets are required. Machine learning and multivariate statistics include many methods suitable for big data. A literature review identified a number of promising methods, and the current status for the use of machine learning for analysis of gas hydrates and FT-ICR MS data was analysed. The literature study revealed that although many studies have used machine learning to predict thermodynamic properties of gas hydrates, very little work have been done in analysing gas hydrate related samples measured by FT-ICR MS. In order to aid their identification, a successive accumulation procedure for increasing the concentrations of hydrate active components was developed by SINTEF. Comparison of the mass spectra from spiked and unspiked samples revealed some peaks that increased in intensity over the spiking levels. Several classification methods were used in combination with variable selection, and peaks related to hydrate formation were identified. The corresponding molecular formulas were determined, and the peaks were assumed to be related to asphaltenes, naphthenes and polyethylene glycol. To aid the characterisation of the oils, infrared spectroscopy (both Fourier Transform infrared and near infrared) was combined with FT-ICR MS in a multiblock analysis to predict the density of crude oils. Two different strategies for data fusion were attempted, and sequential fusion of the blocks achieved the highest prediction accuracy both before and after reducing the dimensions of the data sets by variable selection. As crude oils have such complex matrixes, samples are often very different, and many methods are not able to handle high degrees of variations or non-linearities between the samples. Hierarchical cluster-based partial least squares regression (HC-PLSR) clusters the data and builds local models within each cluster. HC-PLSR can thus handle non-linearities between clusters, but as PLSR is a linear model the data is still required to be locally linear. HC-PLSR was therefore expanded into deep learning (HC-CNN and HC-RNN) and SVR (HC-SVR). The deep learning-based models outperformed HC-PLSR for a data set predicting average molecular weights from hydrolysed raw materials. The analysis of the FT-ICR MS spectra revealed that the large amounts of information contained in the data (due to the high resolution) can disturb the predictive models, but the use of variable selection counteracts this effect. Several methods from machine learning and multivariate statistics were proven valuable for prediction of various parameters from FT-ICR MS using both classification and regression methods.Gasshydrater er et av hovedproblemene for Flow assurance i olje- og gassnæringen ettersom at de kan forårsake blokkeringer i oljerørledninger og prosessutstyr som krever at systemet må stenges ned. Tidligere studier har vist at noen råoljer danner hydrater som ikke agglomererer eller avsetter, men som forblir som transporterbare dispersjoner. Dette antas å være på grunn av naturlig forekommende komponenter til stede i råoljen, men til tross for årevis med forskning er deres nøyaktige strukturer enda ikke bestemt i detalj. Noen studier har indikert at disse komponentene kan stamme fra syrefraksjonene i oljen eller være relatert til asfalteninnholdet i oljene. Råoljer er blant verdens mest komplekse organiske blandinger og kan inneholde opptil 100 000 forskjellige bestanddeler, som gjør dem vanskelig å karakterisere ved bruk av tradisjonelle massespektrometre. Den høye masseoppløsningen Fourier-transform ion syklotron resonans massespektrometri (FT-ICR MS) gir en høyere oppløsning enn tradisjonelle teknikker, som gjør FT-ICR MS i stand til å karakterisere råoljer i større grad og muligens identifisere hydrataktive komponenter. FT-ICR MS spektre inneholder vanligvis titusenvis av topper, og det er nødvendig å bruke databehandlingsmetoder i stand til å håndtere store datasett, med muligheter til å finne underliggende forhold for å analysere spektrene. Maskinlæring og multivariat statistikk har mange metoder som er passende for store datasett. En litteratur studie identifiserte flere metoder og den nåværende statusen for bruken av maskinlæring for analyse av gasshydrater og FT-ICR MS data. Litteraturstudien viste at selv om mange studier har brukt maskinlæring til å predikere termodynamiske egenskaper for gasshydrater, har lite arbeid blitt gjort med å analysere gasshydrat relaterte prøver målt med FT-ICR MS. For å bistå identifikasjonen ble en suksessiv akkumuleringsprosedyre for å øke konsentrasjonene av hydrataktive komponenter utviklet av SINTEF. Sammenligninger av massespektrene fra spikede og uspikede prøver viste at noen topper økte sammen med spikingnivåene. Flere klassifikasjonsmetoder ble brukt i kombinasjon med ariabelseleksjon for å identifisere topper relatert til hydratformasjon. Molekylformler ble bestemt og toppene ble antatt å være relatert til asfaltener, naftener og polyetylenglykol. For å bistå karakteriseringen av oljene ble infrarød spektroskopi inkludert med FT-ICR MS i en multiblokk analyse for å predikere tettheten til råoljene. To forskjellige strategier for datafusjonering ble testet og sekvensiell fusjonering av blokkene oppnådde den høyeste prediksjonsnøyaktigheten både før og etter reduksjon av datasettene med bruk av variabelseleksjon. Ettersom råoljer har så kompleks sammensetning, er prøvene ofte veldig forskjellige og mange metoder er ikke egnet for å håndtere store variasjoner eller ikke-lineariteter mellom prøvene. Hierarchical cluster-based partial least squares regression (HCPLSR) grupperer dataene og lager lokale modeller for hver gruppe. HC-PLSR kan dermed håndtere ikke-lineariteter mellom gruppene, men siden PLSR er en lokal modell må dataene fortsatt være lokalt lineære. HC-PLSR ble derfor utvidet til convolutional neural networks (HC-CNN) og recurrent neural networks (HC-RNN) og support vector regression (HC-SVR). Disse dyp læring metodene utkonkurrerte HC-PLSR for et datasett som predikerte gjennomsnittlig molekylvekt fra hydrolyserte råmaterialer. Analysen av FT-ICR MS spektre viste at spektrene inneholder veldig mye informasjon. Disse store mengdene med data kan forstyrre prediksjonsmodeller, men bruken av variabelseleksjon motvirket denne effekten. Flere metoder fra maskinlæring og multivariat statistikk har blitt vist å være nyttige for prediksjon av flere parametere from FT-ICR MS data ved bruk av både klassifisering og regresjon

    Leaving it all on the Field? Analysing the Appropriate Scope of Vicarious Liability for Athletes in the Sports Industry

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    This research seeks to address the existing uncertainty surrounding the appropriate theoretical justification for vicarious liability, and it does so by developing and deploying a contextual-pluralist model of employer liability. In recognising that none of the purported rationales for the doctrine are entirely satisfactory on their own, this thesis presents a novel framework of vicarious liability that is sensitive to context, factual nuance and empirical insight. In order to test how this model works in practice, this study examines how it might be applied to the eclectic and ever-developing context of sport. This is the first work to provide an in-depth analysis of vicarious liability in the sporting industry, and it draws upon a variety of different sports at numerous levels of expertise. As such, it explores how a more theoretically informed model of vicarious liability might lead to some amateur sports clubs being held strictly liable for the torts of their players. Similarly, it also demonstrates how other entities in professional sport – such as national governing bodies and competition organisers – may also be held vicariously liable for the wrongful behaviour of athletes that compete under their auspices. Furthermore, and in assessing what functions are inherent in an athlete’s employment, this thesis additionally analyses the extent to which sporting employers should be held liable for a variety of personal and non-personal on-the-field injuries. In recognising the broader off-the-field responsibilities of many professional athletes, this study also discusses whether clubs and governing bodies ought to be held vicariously liable for sexual violence committed by their star athletes. In applying my theoretical and socio-legal model of liability to these various contexts, this thesis aims to illustrate what broader lessons we might learn about vicarious liability from a sport-specific analysis. It also seeks to highlight some practical recommendations for sporting stakeholders to implement

    The Typological Diversity of Morphomes: A Cross-Linguistic Study of Unnatural Morphology

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    This is the first typologically-oriented book-length treatment of morphomes, systematic morphological identities, usually within inflectional paradigms, that do not map onto syntactic or semantic natural classes. In the first half of the book, Borja Herce outlines the theoretical and empirical challenges associated with the identification and definition of morphomes, and surveys their links with related notions such as syncretism, homophony, segmentation, and economy, among others. He also presents the different ways in which morphomic structures in a language have been observed to emerge, change, and disappear. The second part of the book contains its core contribution: a database of 120 morphomes across 79 languages from a range of families, which are presented and analysed in detail. A range of findings emerge as a result, including the idiosyncratic nature of morphomes in the Romance languages, the existence of cross-linguistically recurrent unnatural patterns, and the preference for more natural structures even among morphomes. The database also allows further explorations of other issues such as the effect of learnability and communicative efficiency on morphological structures, and the lexical and grammatical informativity of morphs and their distribution

    Structured data abstractions and interpretable latent representations for single-cell multimodal genomics

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    Single-cell multimodal genomics involves simultaneous measurement of multiple types of molecular data, such as gene expression, epigenetic marks and protein abundance, in individual cells. This allows for a comprehensive and nuanced understanding of the molecular basis of cellular identity and function. The large volume of data generated by single-cell multimodal genomics experiments requires specialised methods and tools for handling, storing, and analysing it. This work provides contributions on multiple levels. First, it introduces a single-cell multimodal data standard — MuData — designed to facilitate the handling, storage and exchange of multimodal data. MuData provides interfaces that enable transparent access to multimodal annotations as well as data from individual modalities. This data structure has formed the foundation for the multimodal integration framework, which enables complex and composable workflows that can be naturally integrated with existing omics-specific analysis approaches. Joint analysis of multimodal data can be performed using integration methods. In order to enable integration of single-cell data, an improved multi-omics factor analysis model (MOFA+) has been designed and implemented building on the canonical dimensionality reduction approach for multi-omics integration. Inferring later factors that explain variation across multiple modalities of the data, MOFA+ enables the modelling of latent factors with cell group-specific patterns of activity. MOFA+ model has been implemented as part of the respective multi-omics integration framework, and its utility has been extended by software solutions that facilitate interactive model exploration and interpretation. The newly improved model for multi-omics integration of single cells has been applied to the study of gene expression signatures upon targeted gene activation. In a dataset featuring targeted activation of candidate regulators of zygotic genome activation (ZGA) — a crucial transcriptional event in early embryonic development, — modelling expression of both coding and non-coding loci with MOFA+ allowed to rank genes by their potency to activate a ZGA-like transcriptional response. With identification of Patz1, Dppa2 and Smarca5 as potent inducers of ZGA-like transcription in mouse embryonic stem cells, these findings have contributed to the understanding of molecular mechanisms behind ZGA and laid the foundation for future research of ZGA in vivo. In summary, this work’s contributions include the development of data handling and integration methods as well as new biological insights that arose from applying these methods to studying gene expression regulation in early development. This highlights how single-cell multimodal genomics can aid to generate valuable insights into complex biological systems
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