6,742 research outputs found

    Inter-individual variation of the human epigenome & applications

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    Undergraduate Catalog of Studies, 2023-2024

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    Genome editing of candidate genes related to disease resistance to Piscirickettsia salmonis in Atlantic salmon (Salmo salar)

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    Salmon Rickettsial Syndrome (SRS), caused by the bacterium Piscirickettsia salmonis, is one of the most severe infectious diseases threatening the Chilean Atlantic salmon industry. Among the leading causes of mortality and morbidity, SRS significantly affect the seawater production stage, where biomass losses account for a major economic impact. One potential avenue to tackle SRS is the improvement of host resistance using selective breeding. To accomplish this, insight into the genetic basis of host response, identifying specific genes and pathways involved in this response, and comprehending the potential function these genes have in infection overcome, is valuable. Consequently, this study aims to identify functional genes and pathways that contribute to genetic host resistance to SRS and investigate the effect of CRISPR/Cas9 knockout on these genes during P.salmonis infection. Candidate genes were identified from a previous in vivo large-scale infection study of 2,265 Atlantic salmon smolts injected with P.salmonis and genotyped. These data were used to estimate SRS resistance breeding values. Head-kidney and liver samples for RNA-Seq were obtained from 48 individuals at pre-infection, 3 and 9 days post-infection, and tests of differential expression between pre- and post-infection, and between high and low resistance breeding values were performed. From the thousands of differentially expressed genes, enrichment of several KEGG pathways related to immune response such as bacterial internalisation, intracellular trafficking, apoptosis, and inflammasome was observed in both tissues in fish relatively more resistant to infection. A literature review of the biological function of genes in these pathways highlighted the most suitable candidates for functional studies. Subsequently, five genes related to SRS resistance were successfully edited using a CRISPR/Cas9 Ribonucleoprotein (RNP) transfection to knockout these genes in an Atlantic salmon cell line (SHK-1). An in vitro infection challenge model of the knockout and control cell lines with P.salmonis was performed to elucidate the impact on cytopathic damage, cell viability and bacterial load during infection. These findings suggest a promising avenue of research into the genetic architecture of host resistance to SRS

    The interplay between Natural Killer cells and Pancreatic Stellate cells in Pancreatic Ductal Adenocarcinoma

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    Pancreatic ductal adenocarcinoma (PDAC) is a disease with dismal prognosis. With five-year survival rates of less than 11%, PDAC is set to become the second leading cause of cancer related deaths by 2040. The role of pancreatic stellate cells in pancreatic ductal adenocarcinoma has been well established. However, to date, little remains know about the interaction between these crucial stromal cells and the innate lymphocytes, natural killer (NK) cells, in PDAC. Herein we demonstrate that naïve NK cells possess the functional efficacy to target and kill both quiescent (qPSC) and activated (aPSC) pancreatic stellate cells. Furthermore, qPSC, but not aPSC education of NK cells resulted in decreased NK cell-mediated cancer cell cytotoxicity. NK-PSC direct co-culture was found to modulate both PSC and NK phenotype, as well as functional changes within NK cells, an effect not observed with TranswellTM separation. Multiplex Luminex ELISA further revealed upregulation of IFN-γ and related chemokines in NK cells co-cultured with PSC (activated/quiescent), suggesting that this pathway may be involved in phenotypic modulation. Through global proteomic analysis we demonstrate NK cell-induced differential protein changes in aPSC versus qPSC. Furthermore, we demonstrate changes in intracellular NK pathways as a result of direct contact with PSCs, indicating a dynamic, bidirectional interaction between these two key players. Using multiplex immunohistochemical analysis, we demonstrate that NK cell proximity to CAFs, and not total NK cell infiltrate is correlated with overall survival in PDAC. Consequently, we suggest that the spatial biology of NK/CAFs may play a prognostic role in PDAC and may potentially be used as a tool for patient stratification Taken together, our results demonstrate a significant bidirectional relationship between NK cells and PSC/CAFs in the context of PDAC, providing novel insight into this crucial cell-cell interaction

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

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    Clinical, immunological and genetic features of histiocytic disorders

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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/

    It doesn't end with closure:Optimizing health care throughout life after esophageal atresia repair

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    It doesn't end with closure:Optimizing health care throughout life after esophageal atresia repair

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    UMSL Bulletin 2023-2024

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    The 2023-2024 Bulletin and Course Catalog for the University of Missouri St. Louis.https://irl.umsl.edu/bulletin/1088/thumbnail.jp
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