9,706 research outputs found

    Variations in language use:The influence of linguistic and social factors

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    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/

    Dissecting structural and biochemical features of DNA methyltransferase 1

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    DNA methylation is an epigenetic modification found in every branch of life. An essential enzyme for the maintenance of DNA methylation patterns in mammals is DNA methyltransferase 1 (DNMT1). Its recruitment is regulated through its large N-terminus, which contains six annotated domains. Although most of these have been assigned a function, we are still lacking a holistic understanding of the enzyme's spatio-temporal regulation. Interestingly, a large segment of the N-terminus is devoid of any known domain and appears to be disordered in its sequence. Over the past years, such disordered sequences have increasingly gained attention, due to their role in forming biomolecular condensates through liquid-liquid phase separation (LLPS). These liquid compartments offer specific environmental conditions distinct from the surrounding that can enhance protein recruitment and function. In this work, we explore a potential role for the intrinsically disordered domain (IDR) in the recruitment of DNMT1. Taking an evolutionary approach, we uncover that structural features of the region that are key for IDR function are highly conserved. Moreover, we find conserved biochemical signatures compatible with a role in LLPS. Using a reconstitution assay and an opto-genetic approach in cells, we for the first time show that the DNMT1 IDR is capable of undergoing LLPS in vitro and in vivo. In addition, we define a novel region of interest (ROI) of about 120 amino acids in the IDR that appears to have been inserted in the ancestor of eutherian mammals. Although the ROI has a distinct biochemical signature, we find no effect on the LLPS behavior of the IDR. Therefore, we discuss other potential roles of the ROI related to DNA methylation, for example, imprinting. Finally, we lay the foundation for investigating a biological function of the IDR and establish a system for screening DNMT1 mutant phenotypes in mouse embryonic stem cells. Swift depletion of the endogenous protein is enabled by degron-mediated degradation, while our optimized construct design and efficient derivation strategy ensure the robust expression of the large transgenes. In combination with different methods for DNA methylation read-out, this system can now be used to study the role of the IDR and ROI in maintaining the steady-state level of DNA methylation against mechanisms of passive and active demethylation, but also for studying phenotypes affecting the efficiency of DNMT1 recruitment in the future

    Developing an fMRI paradigm for studying reinforcement learning with gustatory stimuli

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    One of the main challenges for global public health in the modern world is the rising prevalence of obesity. Obtaining a better understanding of the dysregulated feeding behaviour that leads to obesity, by investigating the decision making and learning processes underlying it, could advance our capabilities in battling the obesity epidemic. Consequently, our aim in this study is to design an experiment that could evaluate these processes. We examined ten healthy participants using a modified version of the "probabilistic selection task". We used gustatory stimuli as a replacement for monetary rewards, to assess the effect of nutritional rewards on the learning behaviour. We subsequently analysed the behavioural results with computational modelling and combined this with imaging data simultaneously acquired with a functional magnetic resonance imaging (fMRI) multiband sequence. All participants in this study succeeded in interpreting and interacting with the gustatory stimuli appropriately. Performance on the task was affected by the subjective valuation of the reward. Participants whose motivation to drink the reward and liking of its taste decreased during the task presented difficulties correctly choosing the more rewarding cues. Computational modelling of the behaviour found that the so-called asymmetric learning model, in which positive and negative reinforcement are differently weighted, best explained the group. The acquired fMRI data was suboptimal and we did not detect the neurological activity we expected in the reward system, which is central to our scientific question. Thus, our study shows it is possible to implement the PST with gustatory stimuli. However, to evaluate the corresponding neurological activity, our fMRI configuration requires improvement. An optimised system could be used in further studies to improve our understanding of the neurobiological mechanisms of learning that lead to obesity and elucidate the role of food as a distinctive reinforcer

    LIPIcs, Volume 251, ITCS 2023, Complete Volume

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    LIPIcs, Volume 251, ITCS 2023, Complete Volum

    Scalable Exploration of Complex Objects and Environments Beyond Plain Visual Replication​

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    Digital multimedia content and presentation means are rapidly increasing their sophistication and are now capable of describing detailed representations of the physical world. 3D exploration experiences allow people to appreciate, understand and interact with intrinsically virtual objects. Communicating information on objects requires the ability to explore them under different angles, as well as to mix highly photorealistic or illustrative presentations of the object themselves with additional data that provides additional insights on these objects, typically represented in the form of annotations. Effectively providing these capabilities requires the solution of important problems in visualization and user interaction. In this thesis, I studied these problems in the cultural heritage-computing-domain, focusing on the very common and important special case of mostly planar, but visually, geometrically, and semantically rich objects. These could be generally roughly flat objects with a standard frontal viewing direction (e.g., paintings, inscriptions, bas-reliefs), as well as visualizations of fully 3D objects from a particular point of views (e.g., canonical views of buildings or statues). Selecting a precise application domain and a specific presentation mode allowed me to concentrate on the well defined use-case of the exploration of annotated relightable stratigraphic models (in particular, for local and remote museum presentation). My main results and contributions to the state of the art have been a novel technique for interactively controlling visualization lenses while automatically maintaining good focus-and-context parameters, a novel approach for avoiding clutter in an annotated model and for guiding users towards interesting areas, and a method for structuring audio-visual object annotations into a graph and for using that graph to improve guidance and support storytelling and automated tours. We demonstrated the effectiveness and potential of our techniques by performing interactive exploration sessions on various screen sizes and types ranging from desktop devices to large-screen displays for a walk-up-and-use museum installation. KEYWORDS - Computer Graphics, Human-Computer Interaction, Interactive Lenses, Focus-and-Context, Annotated Models, Cultural Heritage Computing

    DATA AUGMENTATION FOR SYNTHETIC APERTURE RADAR USING ALPHA BLENDING AND DEEP LAYER TRAINING

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    Human-based object detection in synthetic aperture RADAR (SAR) imagery is complex and technical, laboriously slow but time critical—the perfect application for machine learning (ML). Training an ML network for object detection requires very large image datasets with imbedded objects that are accurately and precisely labeled. Unfortunately, no such SAR datasets exist. Therefore, this paper proposes a method to synthesize wide field of view (FOV) SAR images by combining two existing datasets: SAMPLE, which is composed of both real and synthetic single-object chips, and MSTAR Clutter, which is composed of real wide-FOV SAR images. Synthetic objects are extracted from SAMPLE using threshold-based segmentation before being alpha-blended onto patches from MSTAR Clutter. To validate the novel synthesis method, individual object chips are created and classified using a simple convolutional neural network (CNN); testing is performed against the measured SAMPLE subset. A novel technique is also developed to investigate training activity in deep layers. The proposed data augmentation technique produces a 17% increase in the accuracy of measured SAR image classification. This improvement shows that any residual artifacts from segmentation and blending do not negatively affect ML, which is promising for future use in wide-area SAR synthesis.Outstanding ThesisMajor, United States Air ForceApproved for public release. Distribution is unlimited

    Exploring cognitive mechanisms involved in self-face recognition

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    Due to the own face being a significant stimulus that is critical to one’s identity, the own face is suggested to be processed in a quantitatively different (i.e., faster and better recognition) and qualitatively different (i.e., processed in a more featural manner) manner compared to other faces. This thesis further explored the cognitive mechanisms (perceptual and attentional systems) involved in the processing of the own face. Chapter 2 explored the role of holistic and featural processing involved in the processing of self-face (and other faces) with eye-tracking measures in a passive-viewing paradigm and a face identification task. In the passive-viewing paradigm, the own face was sampled in a more featural manner compared to other faces whereas when asked to identify faces, all faces were sampled in a more holistic manner. Chapter 3 further explored the role of holistic and featural processing in the identification of the own face using the three standard measures of holistic face processing: The face inversion task, the composite face task, and the part-whole task. Compared to other faces, individuals showed a smaller “holistic interference” by a task irrelevant bottom half for the own face in the composite face task and a stronger feature advantage for the own face, but inversion impaired the identification of all faces. These findings suggest that self-face is processed in a more featural manner, but the findings do not deny the role of holistic processing. The final experimental chapter, Chapter 4, explored the modulation effects of cultural differences in one’s self-concept (i.e., independent vs. interdependent self-concept) and a negative self-concept (i.e., depressive traits) on the attentional prioritization for the own face with a visual search paradigm. Findings showed that the attentional prioritization for the own face over an unfamiliar face is not modulated by cultural differences of one’s self-concept nor one’s level of depressive traits, and individuals showed no difference in the attentional prioritization for both the own face and friend’s face, demonstrating no processing advantage for the own face over a personally familiar face. These findings suggests that the attentional prioritization for the own face is better explained by a familiar face advantage. Altogether, the findings of this thesis suggest that the own face is processed qualitatively different compared to both personally familiar and unfamiliar face, with the own face being processed in a more featural manner. However, in terms of quantitative differences, the self-face is processed differently compared to an unfamiliar face, but not to a familiar face. Although the specific face processing strategies for the own face may be due to the distinct visual experience that one has with their face, the attentional prioritization of the own face is however, better explained by a familiar face advantage rather than a self-specificity effect

    Effects of language background on executive function: Transfer across task and modality

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    The relation between linguistic experience and cognitive function has been of great interest, but recent investigations of this question have produced widely disparate results, ranging from proposals for a “bilingual advantage,” to a “bilingual disadvantage,” to claims of no difference at all as a function of language. There are many possible sources for this lack of consensus, including the heterogeneity of bilingual populations, and the choice of different tasks and implementations across labs. We propose that another reason for this inconsistency is the task demands of transferring from linguistic experience to laboratory tasks can differ greatly as the task is modified. In this study, we show that task modality (visual, audio, and orthographic) can yield different patterns of performance between monolingual and multilingual participants. The very same task can show similarities or differences in performance, as a function of modality. In turn, this may be explained by the distance of transfer – how close (or far) the laboratory task is to the day to day lived experience of language usage. We suggest that embodiment may provide a useful framework for thinking about task transfer by helping to define the processes of linguistic production and comprehension in ways that are easily connected to task manipulations
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