7,979 research outputs found

    Graduate Catalog of Studies, 2023-2024

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

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    Radiotherapy dosimetry with ultrasound contrast agents

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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/

    Broadband Coherent Anti-Stokes Raman Spectroscopy: A Comprehensive Approach to Analyzing Crystalline Materials

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    Broadband Coherent Anti-Stokes Raman scattering (B-CARS) is an advanced Raman spectroscopy technique used to investigate the vibrational properties of materials. B-CARS combines the spectral sensitivity of spontaneous Raman scattering with the enhanced signal intensity of coherent Raman techniques. While B-CARS has been successfully applied in biomedicine for ultra-fast imaging of biological tissue, its potential in solid-state physics remains largely unexplored. This work delves into the challenges and adaptations necessary to apply B-CARS to crystalline materials and shows its potential as a powerful tool for high-speed, hyperspectral investigations. The theoretical part of this work covers inelastic light-matter scattering fundamentals and the signal generation process of B-CARS, with special attention given to the so-called Non-Resonant Background (NRB). This sample-unspecific signal amplifies the B-CARS intensity but also distorts the shape and position of the measured spectral peaks. A reliable NRB correction becomes crucial to retrieve precise spectral parameters containing information on the investigated material's crystallographic structure, defect density, and stress distribution. The first results chapter presents a practical guideline for an optimized workflow of sample preparation, measurement procedure, and data analysis. The influences of sample surfaces, focus positioning, and polarization sensitivity are discussed. The successful NRB removal is achieved by adapting an algorithm initially designed for biomedical purposes. The second chapter involves a transnational Round Robin investigating the same set of materials using different experimental setups. The influences of laser source, detection range, and transmission vs. epi detection are explored to optimize the experimental parameters. This work showcases applications such as high-speed, hyperspectral imaging of ferroelectric domain walls in LiNbO3, demonstrating the potential of B-CARS in the cutting-edge field of domain wall engineering. Additionally, imaging and polarization-sensitive measurements are shown for MoO3 flakes, paving the way for B-CARS investigations of 2D materials. The final chapter presents advanced techniques, such as Three-Color CARS and Time-Delay CARS, applied to crystalline materials. Three-Color CARS is especially promising, as it enhances the signal intensity for low-frequency Raman modes, which are particularly interesting for solid-state physics compared to the usual large-shift modes investigated in biomedical research. Meanwhile, Time-Delay CARS is sensitive to relaxation processes of vibrational and NRB states, enabling experimental NRB removal and lifetime measurements. Additionally, a neural network-based NRB removal method is presented, eliminating the need for a prior NRB spectrum and offering rapid computation. In summary, this work demonstrates the successful implementation of B-CARS for crystalline materials and provides a comprehensive guideline for the optimal experimental setup, workflow, and data processing. The application of B-CARS for imaging bulk crystalline materials, ferroelectric domain walls, and 2D structures shows promising possibilities for future research

    Graduate Catalog of Studies, 2023-2024

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    The role of the oral microbiome in the immunobullous diseases pemphigus vulgaris and mucous membrane pemphigoid and oral lichen planus

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    Saliva is formed from contributions of salivary glands and the serum exudates principally from gingival margins or damaged mucosa combined with components derived from the environment, including a community of microorganisms - the microbiome. I postulate that changes in microbial diversity and population structure play key roles in the modulation of host- microbial interactions which influence both the hypersensitive autoimmune responses and inflammation seen in these inflammatory mucocutaneous disorders. For my research, a total of 186 participants were recruited: 48 mucous membrane pemphigoid (MMP), 48 pemphigus vulgaris (PV), 50 oral lichen planus (OLP) patients, and 40 healthy controls. Unstimulated whole saliva, subgingival plaque, serum, and plasma samples were collected from 186 participants. In addition, metadata were collected on the following covariates: age, gender, ethnicity, type of the diet, disease history and therapeutic intervention in the preceding six months. Oral disease severity scores (ODSS) were assessed, and periodontal status was examined using a periodontal six pocket chart. To characterise microbiome profiles, saliva and subgingival plaque were processed for sequencing genomic DNA using the NGS Shotgun metagenomics sequencing technique. Inflammatory cytokines and proteases were investigated in saliva and serum using Human Magnetic Luminex Screening Assay (R&amp;D Systems). Selected cytokines were analysed by enzyme-linked immunosorbent assay (ELISA) technique (R&amp;D Systems) to determine host inflammatory responses in saliva and serum samples. Additionally, saliva and plasma samples were analysed for metabolites by nuclear magnetic resonance (NMR). Significant increases in periodontal score (PISA) in all three groups of disease were identified compared to healthy control group with significant positive correlation between oral disease severity (ODSS) and PISA in OLP and PV groups. All three groups of diseases had significantly higher levels of inflammatory Th2/Th17 cytokines (IL-6, IL-13 and IL-17 in saliva samples), as well as higher levels of MMP-3 matrixins in saliva. In addition, there were positive correlations between ODSS and salivary IL-6, IL-13 and MMP-3 in saliva of OLP, salivary and serum levels of IL-6 and MMP-3 in MMP group, and significant association of salivary IL-6, IL-1β and MMP-3 in PV group. Metabolomic data showed that saliva is a better biofluid for correlation of the metabolomic profile with oral disease severity than plasma. Salivary ethanol was corelated with disease severity in the OLP group, whereas in PV was a strong correlation of ODSS with choline. Finally, a unique microbial community was found in each group of diseases. In the MMP group, ODSS was significantly correlated with L. hofstadii, C. sputigena, N. meningitidis, N. cinerea and P. sacchar0lytica. In PV, a positive correlation was found with F. nucleatum, G. morbillorum, and E. corrodens, G. elegans, H. sapiens and T. vincentii. In OLP, the disease tends to worsen when there was reduced abundance of X. cellulosilytica, Actinomyces ICM 47, S. parasanguinis, S. salivarius, L. mirabilis and O. sinus. Lower microbial diversity was correlated with ODSS in saliva and plaque of the OLP group. In conclusion, this study provides strong evidence of the complex interplay between the oral microbiome, immunological factors, and metabolites in the context of immunobullous diseases and OLP. The findings highlight the integral role of oral bacteria in disease progression, the significance of immune dysregulation, and the potential impact of specific microbial species and metabolic pathways. These insights give the way for further research and clinical applications, offering the promise of personalized approaches for diagnosis, and management of OLP, MMP and PV. Future investigations should focus on discovering the mechanistic details underlying these associations and validating the identified biomarkers in larger patient cohorts, ultimately contributing to a deeper understanding of the pathogenesis of these conditions

    Targeting Strategies to Optimize the Therapeutic Potential of Gold Compounds Against HER2-Positive Breast Cancers

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    The overexpression of HER2 accounts for 20-30% of breast cancer tumors and not only serves as a marker for poor predictive clinical outcomes but also as a target for treatment. Antibody-drug conjugates (ADCs) combine the selectivity of monoclonal antibodies (mAbs) with the efficacy of chemotherapeutic drugs to provide targeted treatment without toxicity to normal tissue. Most of the ADCs currently in the clinic for cancer chemotherapy are based on complex organic molecules. In contrast, the conjugation of metallodrugs to mAbs has been overlooked when there is enormous potential in this area with the resurgence of metal-based drugs as prospective cancer chemotherapeutics. In this study, we have evaluated the efficacy of gold(I)- based cytotoxic payloads in ADCs based on the HER2 targeted mAb, Trastuzumab, Pertuzumab and THIOMAB®. The ADCs were selective and highly efficacious towards HER2-positive breast cancer cells. We then assessed the anti-tumor efficacy of a selected gold-based ADC in a HER2- positive tumor-bearing mouse model and found the ADCs to be exceedingly potent in reducing tumor size by 81% with little to no toxicity in the animals. In addition to this, we aimed to develop a method of drug delivery combining liposomal formulations and HER2 targeted mAbs in the form of immunoliposomes to further increase the cytotoxic gold-payload content. These nanocarriers are advantageous in that they have an extended blood circulation time, allowing for higher accumulation in the tumor, they are much more sensitive towards the tumor micro-environment, and they allow for precise control of drug release. We have demonstrated that immunoliposomes containing gold(I)-phosphane species are cytotoxic and selective to HER-2 positive breast cancer cells. The encapsulation of a gold compound containing a fluorescent phosphane, allowed us to study the localization of the free vs. encapsulated drug by confocal microscopy. These studies showed that while the free drug localizes to lysosomes to be sequestered and degraded, the encapsulated and targeted drug accumulated more inside the cells and localized to the mitochondria and ER, indicating that protection by liposomal vesicles prevents early degradation of the compound and better internalization. Taken together, the development of gold- based ADCs and immunoliposomes with specific targeting and cytotoxic abilities exhibit tremendous potential in the field of metal-based drugs and nanocarrier systems and may be highly relevant for clinical translation

    Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

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    Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

    LIPIcs, Volume 251, ITCS 2023, Complete Volume

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    LIPIcs, Volume 251, ITCS 2023, Complete Volum
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