Browsing by Subject "Transcriptomics"

Now showing 1 - 10 of 10
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    Application of Systems Biology Analysis to Hepatic Injury Following Hemorrhagic Shock
    (2014-05) Determan, Charles Edward Jr
    Introduction: This dissertation is focused on the metabolomic and transcriptomic changes that occur as a result of carbohydrate prefeeding during hemorrhagic shock and trauma within the liver of a porcine model. The risk of trauma and hemorrhagic shock continues to be an important issue in both military and civilian sectors. As such, we explored the impact of a prior fed state upon the overall response to hemorrhagic shock and resuscitation. The primary hypotheses were that changes in metabolism at the metabolomic and transcriptomic levels would be dependent upon the fed state. In addition, this thesis explores a more comprehensive analysis of metabolomics datasets to standardize analysis and improve overall consistency.Materials and Methods: Algorithm comparison was accomplished using six commonly applied methods to three synthetic datasets, of different sample sizes, and three openly accessible published datasets. This comparison also incorporated metrics to measure consistency of identified features (i.e. stability) to provide further confidence in results. Metabolomics analysis was accomplished with nuclear magnetic resonance spectroscopy (NMR) and Chenomx software to profile and quantify metabolites in liver extracts. The metabolome was subsequently analyzed with partial least squares discriminant analysis (PLS-DA). Transcriptomics analysis was conducted using next-generation sequencing (NGS) technology to employ RNA-sequencing (RNA-seq) on mRNA extracts from liver biopsies. The RNA-seq data was analyzed using typical processing techniques to generate a count matrix and subsequently analyzed with the Bioconductor package EdgeR. Results: The comparison of algorithms showed that the best algorithm is associated with differently structured datasets (e.g. number of features, number of groups, sample size, etc.). Analysis of the liver metabolome revealed changes in carbon energy sources, amino acid metabolism, oxidative stress, and membrane maintenance. Transcriptomic analysis revealed changes in carbohydrate metabolism, cytokine inflammation, cholesterol synthesis and apoptosis. In addition, there is evidence of increased cytoskeleton reorganization which may correspond to a shrunken, catabolic state which provides and anti-inflammatory condition to mitigate cellular damage.Conclusion: The response to hemorrhagic shock and resuscitation is altered with respect to a fasted or carbohydrate prefed state. Metabolomics and transcriptomic analyses suggest altered metabolic pathways as a result of fed state. Altered carbohydrate metabolism was readily identified thereby confirming both methods were successful. Additionally, indications of membrane maintenance that follow cytoskeletal remodeling and cellular shrinkage are potentially reflected by 3-Hydroxyisovalerate and sn-Glycero-3-phosphocholine. These results provide further evidence for pre-conditioning (e.g. altered diet) and hypertonic resuscitation methods to possibly improve patient outcome. Further research is required in alternative prefeeding substrates (e.g. protein, lipid, etc.) as well as improving the integration of different systems level datasets to understand more thoroughly the systemic effects of hemorrhagic shock and resuscitation.
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    Characterization of Natural Killer Cell Activation and Functionality for Cell Therapy Applications
    (2020-12) One, Jennifer
    Natural killer (NK) cells are a promising emerging allogeneic cell therapy due to their cytotoxic effector and cytokine producing functions and lack of induction of Graft-vs-Host Disease. For allogeneic clinical applications, NK cells isolated from a single donor must be expanded through an efficient large-scale biomanufacturing process to treat many patients and produce an economical off-the-shelf therapy. Critically, the cultured NK cells must maintain functionality post-expansion to be an effective cellular therapy. Through multiple rounds of activation with K562 artificial antigen-presenting cells (aAPCs), the quantity of NK cells can expand by several orders of magnitude. However, the changes that chronic stimulation might induce in cell cycle status, metabolism, and ex vivo functionality of NK cells are not well understood. Even less is known regarding how changes in these cell characteristics may influence their in vivo functionality. In this work, we conducted a systematic evaluation of the activation and expansion process of NK cells through transcriptome analysis, dynamics of chromatin accessibility, metabolic characterization, and phenotypic analysis of exhaustion and senescence over time. By understanding the transcriptional and epigenetic signature of the K562-activated NK cells, we have identified potential genes and transcription factors that may regulate K562 activation in order to develop a bioprocess that phases out these feeder cells from the culture process. Furthermore, rigorous characterization of NK cell growth and receptor expression during expansion revealed that prolonged stimulation results in an immature, exhausted, cytokine-producing phenotype over time. Changes in NK growth kinetics corresponds to shifts in NK cytotoxicity in select target cancer cell lines, indicating proliferative potential may be an indicator of a good donor for clinical use. Collectively, understanding the effects of activation and consequent proliferation of NK cells would remove a major roadblock in the biomanufacturing of NK cells, thus laying the groundwork for their potential use as an off-the-shelf allogeneic cellular therapy. Insights into mechanisms underlying activation and expansion provide a path to develop strategies to eliminate K562 aAPCs altogether, which would be desirable from a regulatory standpoint and further ease the transition from benchtop to biomanufacturing as well as improve efficacy in the clinic.
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    Data for: Using the Daphnia magna Transcriptome to Distinguish Water Source: Wetland and Stormwater Case Studies
    (2022-07-18) Jankowski, Mark D; Fairbairn, David J; Baller, Joshua A; Westerhoff, Benjamin M; Schoenfuss, Heiko L; jankowski.mark@epa.gov; Jankowski, Mark
    A major challenge in ecotoxicology is accurately and sufficiently measuring chemical exposures and biological effects given the presence of complex and dynamic contaminant mixtures in surface waters. Our study examined the performance of the Daphnia magna transcriptome to detect distinct responses across three water sources in Minnesota: laboratory [well] waters, wetland waters, or stormwaters. Pyriproxyfen (PPF) was included as a gene expression and male neonate production positive control to examine whether gene expression resulting from exposure to this well-studied juvenoid hormone analog can be detected in complex matrices. Laboratory-reared (<24 hr) D. magna were exposed to a water source and/or PPF for 16 d to monitor phenotypic changes or 96 hr to examine gene expression responses using Illumina HiSeq 2500 (10 million reads per library, 50-bp paired-end (2x50)).
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    Genomic and transcriptomic approaches for the advancement of CHO cell bioprocessing
    (2014-06) Vishwanathan, Nandita
    Recombinant protein therapeutics have transformed healthcare by paving the way for the treatment of refractory illnesses like cancer and arthritis. Chinese hamster ovary (CHO) cells are the major workhorse for the production of these therapeutics. Striving for continual improvements in the productivity and quality of protein produced in CHO cells, many process enhancements have been successfully implemented. However, many processes are still empirical, and we have little understanding of the mechanisms for these methods. The availability of genomic resources for CHO cells has ushered in a `genomics' era in bioprocessing. Genomic resources can now be employed to understand and improve cell lines and processes to enhance the productivity and quality of protein therapeutics produced by CHO cells. Seeking the development of genomic resources for CHO cells, the Chinese hamster genome and transcriptome were sequenced, assembled and annotated. Such transcriptomic resources can be used to study the inherent transcriptomic variability in CHO cells. The genetic cues identified from the study of the variability in the glycosylation pathway genes opens up several opportunities to manipulate protein quality. The relative expression of isozymes in CHO cells affect metabolic characteristics, which in turn may potentially impact product quality or even process robustness. The comparative study of isozymes can give important clues for cell engineering and process development. The isozyme distribution in CHO cells indicates a very high overall glycolytic rate, insinuating to the possibility of manipulating glycolytic flux for improving processes. Engineering superior metabolism through cell engineering can be used to reduce glycolytic flux in the late stage of the fed batch culture to reduce lactate accumulation. A novel dynamic promoter was used to drive the expression of a fructose transporter selectively in the late stages of the culture. By maintaining adequately low fructose levels in the late stage, the glycolytic flux was reduced significantly to induce lactate consumption. Since lactate accumulation is well accepted to be detrimental to productivity, this phenotype is desired for bioprocessing. In addition to such high productivity processes, high producing cells are also desired. The lengthy process of cell line development transforms non-producing cells to high producers. The molecular changes in this transformation were elucidated by studying the transcriptome of CHO cells during cell line development. We hypothesize that methotrexate treatment not only increases the transgene copy number, but also enriches cells with superior growth, energy metabolism, and secretion capabilities. This leads to an enriched population of high producers. The sustenance of high productivity over several generations depends on the stability of the integration site of the transgene. Two methods for identifying the cell's transgene integration site were developed and optimized. These methods can be applied for high throughput investigation of stability of integration sites.The application of genomics in bioprocessing has sparked a systems approach to investigate genetic regulation. This knowledge paved the way for controlling cellular metabolism and achieve stable and high producing cell lines and processes. Such genome scale analyses have a great potential to advance the capacity of CHO cells for biopharmaceutical applications.
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    Genomics and domestication of Field Pennycress (Thlaspi arvense)
    (2015-05) Dorn, Kevin
    Thlaspi arvense (field pennycress) is a cold tolerant oilseed species that is being domesticated as a new rapid cycling, winter annual cover crop and feedstock for biodiesel production. Pennycress is related to Arabidopsis thaliana, a model species that has provided an in-depth understanding of many basic developmental and physiological plant processes, which will provide vital information for the rapid domestication of a wild species into a new crop. By targeting key pennycress traits for improvement, such as reducing seed dormancy, increasing rates of spring flowering and maturity, increasing yield, and modifying seed oil composition, we are poised to develop a new winter cash crop that can fit within the corn/soybean rotation. To enable a mutation breeding approach that utilizes the massive amount of Arabidopsis-based knowledge, genomic resources are needed to identify target genes believed to influence key traits. In this dissertation, the first comprehensive annotated transcriptome assembly and comparative analyses are presented, along with the first draft genome sequence for pennycress. In these analyses, target assembled transcripts and corresponding DNA sequences are identified and compared to Arabidopsis homologs and enable the forward and reverse genetic screening of large scale mutant populations. An analysis of winter and spring annual pennycress accessions is also presented, which identified several wild alleles of the pennycress FLOWERING LOCUS C homolog which was found to be responsible for differentiating between spring and winter annual phenotypes. The resources presented herein will provide an unprecedented set of tools to enable the rapid domestication of a new crop species.
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    Improved Computer Vision Algorithms for High-Throughput Targeting of Single Cells in Intact Tissue for Automated Microinjections
    (2021-10) O'Brien, Jacob
    Microinjection is a technique for organism-level and cellular-level manipulation of biological systems. The precise nature of microinjection permits the ability to target single cells in intact tissue which has enabled the study of cell-type related phenomena in development and disease progression. We envisioned the use of single-cellular microinjection as a tool for tagging cells with unique oligonucleotide barcodes that can be used during post-injection transcriptomic analysis to relate the transcriptomic reads with originally injected cells. For this process to be viable, we needed a system that was capable of precisely identifying the locations of cells in 3D tissue, assessing their feasibility for injection, and conducting rapid and large-scale microinjection into the identified cells. In this thesis, we report the development of such system. Our automated system uses computer vision algorithms to identify the 3D position of epifluorescent cells in intact tissue slices and assign them a quality metric to prioritize injections. The system guides a robotic micromanipulator to these cells and attempts injections while another computer vision algorithm and Kalman filter are used to improve the robot’s positioning accuracy. Additionally, cell impalement and cell filling detection algorithms were developed to evaluate injection success. We discovered, through a microinjection parameter sweep, an optimum combination of parameters to enable successful microinjection into a variety of cell types and tissue types. We used the optimized parameters to demonstrate automated tagging of single cells with a fluorescently labeled antibody targeting the nuclear pore complex proteins as a precursor step to fluorescence-based nuclei sorting and later transcriptomic analysis.
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    An Interdisciplinary Geochemical and Genomics Approach to Understanding Fungal Selenium Transformations for the Bioremediation of Contaminated Waters
    (2021-07) Sabuda, Mary
    Selenium (Se) is both a micronutrient required for most life and an element of environmental concern due to its toxicity in high concentrations. Se can be released into the environment through both natural and anthropogenic (human) activity, where it can exist as volatile or organic Se(-II), nanoparticulate Se(0), or aqueous Se(+IV/VI). Coal mining, processing, and burning can release high levels of Se to the environment, as selenium can easily substitute for sulfur, a main component of coal. Se is also useful in the medical field, where it has anticancer properties and Se(0) is an effective coating on medical devices. While most knowledge of biotic Se transformations is related to either anaerobic or aerobic bacterial processes, some common soil Ascomycota fungi can reduce Se under oxic conditions. These microeukaryotes readily transform elevated concentrations of this essential toxin from a bioavailable aqueous phase (Se(IV/VI)) to solid or volatile phases (Se(0/-II)), which is ideal for engineering efficient, cost-effective treatment strategies for Se-contaminated environments. Elucidating the geochemical and genetic mechanisms behind filamentous fungal Se transformation strategies will progress biotechnological applications for biogenic Se nanoparticles, and aid in a more complete understanding of Se biogeochemical cycling.
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    Multi-omic analysis of hibernator skeletal muscle and calcium handling regulation
    (2016-05) Anderson, Kyle
    Mammalian hibernation is a strategy employed by many species to survive fluctuations in resource availability and environmental conditions. Hibernating mammals endure conditions of dramatically depressed heart rate, body temperature, and oxygen consumption; yet do not show the typical pathological responses. Because of the high abundance and metabolic cost of skeletal muscle, not only must it adjust to the constraints of hibernation, but it is also positioned to play a more active role in the initiation and maintenance of the hibernation phenotype. My M.S. thesis research has primarily focused on the generation and analysis of two high-throughput ‘omics screens in thirteen-lined ground squirrel skeletal muscle. A transcriptomic analysis using Illumina HiSeq2000 technology identified 1,466 differentially expressed genes throughout their circannual cycle. This RNAseq data allowed for greater protein identifications in an iTRAQ based proteogeomic analysis of the same animals. Of the 1,563 proteins identified by this proteogenomic approach, 232 were differentially expressed. These data support previously reported physiological transitions, while also offering new insight into specific mechanisms of how hibernator muscles might be reducing nitrogenous waste, preserving mass and function, and signaling to other tissues. Sarcolipin is a specific gene of interest that shows a 10-fold difference in expression between hibernation and spring collection points. Because of sarcolipin’s interaction with the SERCA pump and their role in muscle-based thermogenesis and calcium homeostasis bioenergetics, I have developed methods to measure the consequences of this differential expression.
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    Pre-clinical Strategies to Overcome Drug-Resistant Multiple Myeloma: Predictive Transcriptomics and Targeting the Myeloma Epigenome
    (2018-04) Harding, Taylor
    Multiple myeloma remains an incurable hematological malignancy due to the failure of standard-of-care therapies to broadly target a genetically heterogeneous disease and an inability overcome inevitable drug-resistant relapse. This dissertation will address this outstanding problem through two approaches: transcriptomic profiling to predict resistance to proteasome inhibitors and pre-clinical evaluation of epigenetic-targeting therapies to broadly target the myeloma epigenome. First, our goal was to develop a gene expression signature that predicts response specific to proteasome inhibitor (PI) treatment in MM. Using a well-characterized panel of human myeloma cell lines (HMCLs) representing the biological and genetic heterogeneity of MM, we created an in vitro chemosensitivity profile in response to treatment with the four PIs as single-agents. Through gene expression profiling and machine learning-based computational approaches we identified a 42-gene expression signature that could not only distinguish good and poor PI-response in the HMCL panel, but could also be successfully applied to four different clinical datasets on MM patients undergoing PI-based chemotherapy to distinguish between extraordinary (good and poor) outcomes. Our results demonstrate the use of in vitro modeling and machine learning-based approaches to establish predictive biomarkers of response and resistance to drugs that may serve to better direct myeloma patient treatment options. Epigenetic abnormalities are abundantly present in multiple myeloma and accumulating evidence suggests that the histone methyltransferase EZH2 is aberrantly active in MM. We tested the efficacy of EZH2 specific inhibitors in a large panel of human MM cell lines (HMCLs) and found that only a subset of HMCLs demonstrate single agent sensitivity despite ubiquitous global H3K27 demethylation. Pre-treatment with EZH2 inhibitors greatly enhanced the sensitivity of HMCLs to the pan-HDAC inhibitor panobinostat in nearly all cases regardless of single agent EZH2 inhibitor sensitivity. Transcriptomic profiling revealed large-scale transcriptomic alteration by EZH2 inhibition highly enriched for cancer-related pathways. Further analysis demonstrated that combination treatment further perturbed oncogenic pathways and signaling nodes consistent with an antiproliferative/pro-apoptotic state. We conclude that combined inhibition of HDAC and EZH2 inhibitors is a promising therapeutic strategy to broadly target the epigenetic landscape of aggressive MM.
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    A Systems Approach to Studying the Differentiation of Stem Cells Towards Hepatocytes
    (2017-08) Chau, David
    The recent advancements in stem cell biology have allowed for new and exciting opportunities to use stem cells in clinical and industrial applications. Stem cells have the unique ability to self-renew and differentiate into any specialized cell type found in the body. Using certain mechanical and biochemical cues, stem cells can be directed to become any specific cell type, such as hepatocytes. A robust and efficient process for expansion and differentiation to generate large quantities of functional hepatocytes from stem cells will be essential to establishing a stem cell bioprocess in the future for therapeutic and industrial applications of hepatocytes. In this study, a differentiation protocol with soluble growth factors and cytokines was used to mimic the key signaling cues during embryonic development. However, most directed differentiation processes have run into issues with limited scalability and lack of functionality in the differentiated cells. In an effort to bring stem cell therapy closer to reality, our strategy was to use a systems-based approach to enhance the quality and yield of stem cell-derived hepatocytes. To achieve higher cell yield, we modified an existing differentiation protocol to incorporate a cell expansion stage to facilitate simultaneous differentiation and cell growth. Using transcriptome analysis and mass cytometry, we showed how the population of cells changed over time on both the transcript and protein level. Both analyses revealed that with the new expansion stage, we obtained a higher quantity of hepatocytes within the same time frame compared to the conventional method of differentiation. We then showed the capability to scale up our differentiation for larger scale cultures by adapting the expansion stage onto Cytodex 3 microcarriers. Using the same culture volume as a tissue plate culture, we demonstrated the ability to achieve up to a 5-fold increase in cell number with a final cell density in the range of 4-5x106 cells/ml. These strategies show that the demand for large quantities of hepatocytes can be met by translating the conventional method of differentiation to suspension microcarrier differentiation. Encouraged by our ability to yield higher cell density using microcarrier culture, we explored assessing the functional maturity of our stem cell-derived hepatocytes using transcriptome analysis. We showed that stem cell-derived hepatocytes are still clearly different when compared to primary hepatocytes at the transcriptome level. In addition to evaluating cells using transcriptome analysis, we wanted to be able to compare the current in-vitro processes to embryonic liver development to understand the genetic roadblocks. The transcriptome data from hESCs hepatocyte differentiation was integrated with mouse liver development using principal component analysis and batch corrections. This allowed us to create a unified developmental scale to compare samples from different species and in-vitro to in-vivo platforms. The meta-analysis revealed that stem cell-derived hepatocytes are equivalent to the functional maturity of developing cells at E15 in mouse development. From the transcriptome analysis, we observed many different genes in energy metabolism with dynamic behavior over the course of differentiation. We sought to understand the effect of changes in different metabolic genes and the impacts on metabolic transition during differentiation. We characterized the energy metabolism of hESCs and assessed the metabolic demand of cells at different stages of differentiation. hESCs and early differentiated cells exhibited a high glycolytic flux. transitioning towards an oxidative metabolism as the differentiation progressed. Furthermore, using confocal microscopy, we also characterized the activity and morphology of the mitochondria in the cells at different stages of differentiation. Using the consumption rates of different nutrients as an input to our metabolic flux model along with our transcriptome findings, we were able to gain a deeper understanding of the metabolic behavior of cells during differentiation. Our analysis revealed that cells consume lower amounts of glucose over the course of the differentiation but become more efficient at transporting pyruvate into the mitochondria leading to increased oxidative phosphorylation. However, our metabolic and transcriptome data revealed that our stem cell-derived hepatocytes are not capable of mature metabolic functions such as gluconeogenesis, supporting the immature phenotype that has been described in literature. Together, these studies reveal that stem cells can provide a renewable and scalable source of hepatocytes for therapeutic applications. These cells demonstrate some phenotypic and functional properties of primary hepatocytes but have some contrasting elements compared to their in-vivo counterparts that will need further genetic intervention to enhance their maturation before cellular therapy can become a reality. However, this work is invaluable as it contributes to the current status of the field and facilitates the translation of laboratory practices of stem cell culture into a scalable technology.