2,159 research outputs found

    Undergraduate Catalog of Studies, 2023-2024

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    Investigating Human Embryo Implantation – Developing Clinical Applications from in vitro Models

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    Introduction: While assisted conception success rates have increased, factors limiting IVF success include inadequacies in identifying viable embryos, and transfer of embryos into uteri with an unknown state of receptivity. Aims and experimental approaches: The aims of this project are to determine the possibility of using non-invasive techniques to reveal differences between preimplantation human embryos which successfully form a pregnancy and those that fail to implant. The experimental approaches are: 1 Sampling of conditioned media and co-culture with a 3D in vitro model of mid-secretory phase normal human endometrium, followed by transcriptomic analysis of these endometrial cells; 2 Development of a time lapse annotation system to improve selection of PN stage frozen embryos cultured to blastocyst and replaced in FET cycles. Methods: Endometrial epithelial and stromal cells in an in vitro model of mid-secretory phase human endometrium were exposed to conditioned media samples from 10 human embryos cultured singly to the blastocyst stage, with known pregnancy outcomes. These cells were subjected to RNA sequencing and transcriptomic analysis. Time lapse recordings of these embryos were taken through an experimental AI model (eM-Life). Retrospective analysis and annotation of time lapse videos of embryo development of 193 PN stage frozen embryos thawed and cultured to the blastocyst stage for replacement in an FET cycle was performed. Results: Endometrial epithelial cells showed changes in gene expression in response to media from successful embryos, while stromal cells responded to a lesser extent to media from unsuccessful embryos. The deep learning model ranked embryos on morphology but did not correlate with endometrial response in this project. From the analysis of 193 PN stage frozen embryos, statistically significant differences in several morphokinetic parameters between implanting and non-implanting embryos were found and morphological differences not previously studied in frozen thawed embryos relating to embryo viability were identified. Conclusions: Both experimental approaches revealed differences between embryos which implant successfully and those which fail, not detected by standard morphological grading. Further work is needed to identify upstream factors in conditioned media which cause gene expression changes in the in vitro endometrial model, and to test the morphokinetic model developed for frozen embryos in culture

    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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    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/

    Graduate Catalog of Studies, 2023-2024

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    CANCER TREATMENT BY TARGETING HDAC4 TRANSLOCATION INDUCED BY MICROSECOND PULSED ELECTRIC FIELD EXPOSURE: MECHANISTIC INSIGHTS THROUGH KINASES AND PHOSPHATASES

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    Epigenetic modifications, arising from sub-cellular shifts in histone deacetylase (HDAC) activity and localization, present promising strategies for diverse cancer treatments. HDACs, enzymes responsible for post-translational histone modifications, induce these epigenetic changes by removing acetyl groups from ε-N-acetyl-lysine residues on histones, thereby suppressing gene transcription. Within the HDAC group, class IIa HDACs are notable for their responsiveness to extracellular signals, bridging the gap between external stimuli, plasma membrane, and genome through nuclear-cytoplasmic translocation. This localization offers two significant mechanisms for cancer treatment: nuclear accumulation of HDACs represses oncogenic transcription factors, such as myocyte-specific enhancer factor 2C (MEF2C), triggering various cell death pathways. Conversely, cytoplasmic HDAC accumulation acts similarly to HDAC inhibitors by silencing genes. My dissertation introduces an innovative approach for glioblastoma and breast cancer treatment by investigating the application of microsecond pulsed electric fields. It particularly focuses on HDAC4, a class IIa HDAC overexpressed in these cancers. Beyond demonstrating HDAC4 translocation, my research delves into the intricate roles of kinases and phosphatases, shedding light on the underlying factors governing HDAC4 translocation

    Characterisation of peroxisomes in the fission Yeast Schizosaccharomyces pombe and slime mold Dictyostelium discoideum

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    Peroxisome is a compartment that is found in most eukaryotic organisms' cells. It has several crucial roles, such as fatty acid beta (β) oxidation and hydrogen peroxide (H2O2) detoxification. It contains many essential enzymes, including oxidase and catalase, and has several metabolic and non-metabolic pathways, depending on the environment and the organisms within its cells. This study investigates the role of peroxisomes in two organisms, S. pombe and D. discoideum. Although S. pombe is a well-studied yeast, there is only one study of this yeast that has focused on peroxisomes. This study offers a few crucial observations, including that S. pombe contains peroxisomes, that GFP containing a well-characterized PTS1 (SKL) is efficiently imported, and that peroxisome numbers increase in cells grown on a fatty acid as the sole carbon source, suggesting a role for peroxisomes in fatty acid degradation. The starting point in my research was initially a bioinformatics screen. This screening recognized the enzymes imported into peroxisomes based on the presence of a potential peroxisomal targeting signal. A few proteins were found. However, the low number of proteins with a classical PTS might be the result of different targeting signals that are not recognized by our bioinformatics parameters. Indeed, in other organisms, there are proteins without PTS1 that still use Pex5 for import. The first example is S. cerevisiae Acyl-CoA oxidase. In a global yeast two-hybrid screen, S. pombe Pex5 was found to bind S. pombe Str3 and Lys3. Consequently, we think that there is conserved targeting of a peroxisomal protein lacking a PTS1 and PTS2 imported into the peroxisome by Pex5. One of these is the Str3 case. Interestingly, proteins involved in peroxisomal fatty acid β -oxidation are absent from the S. pombe genome, casting doubt on the conclusions from the previous study and explaining the low number of potential peroxisomal enzymes. In D. discoideum, this study investigates the dynamic regulation of peroxisome numbers in response to growth conditions and identifies peroxisomal import and contents through a proximity labeling approach (BioID). Overall, this study sheds light on the roles and regulation of peroxisomes in these two organisms

    Tracking unlabeled cancer cells imaged with low resolution in wide migration chambers via U-NET class-1 probability (pseudofluorescence).

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    Cell migration is a pivotal biological process, whose dysregulation is found in many diseases including inflammation and cancer. Advances in microscopy technologies allow now to study cell migration in vitro, within engineered microenvironments that resemble in vivo conditions. However, to capture an entire 3D migration chamber for extended periods of time and with high temporal resolution, images are generally acquired with low resolution, which poses a challenge for data analysis. Indeed, cell detection and tracking are hampered due to the large pixel size (i.e., cell diameter down to 2 pixels), the possible low signal-to-noise ratio, and distortions in the cell shape due to changes in the z-axis position. Although fluorescent staining can be used to facilitate cell detection, it may alter cell behavior and it may suffer from fluorescence loss over time (photobleaching).Here we describe a protocol that employs an established deep learning method (U-NET), to specifically convert transmitted light (TL) signal from unlabeled cells imaged with low resolution to a fluorescent-like signal (class 1 probability). We demonstrate its application to study cancer cell migration, obtaining a significant improvement in tracking accuracy, while not suffering from photobleaching. This is reflected in the possibility of tracking cells for three-fold longer periods of time. To facilitate the application of the protocol we provide WID-U, an open-source plugin for FIJI and Imaris imaging software, the training dataset used in this paper, and the code to train the network for custom experimental settings
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