2,334 research outputs found

    The development of bioinformatics workflows to explore single-cell multi-omics data from T and B lymphocytes

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    The adaptive immune response is responsible for recognising, containing and eliminating viral infection, and protecting from further reinfection. This antigen-specific response is driven by T and B cells, which recognise antigenic epitopes via highly specific heterodimeric surface receptors, termed T-cell receptors (TCRs) and B cell receptors (BCRs). The theoretical diversity of the receptor repertoire that can be generated via homologous recombination of V, D and J genes is large enough (>1015 unique sequences) that virtually any antigen can be recognised. However, only a subset of these are generated within the human body, and how they succeed in specifically recognising any pathogen(s) and distinguishing these from self-proteins remains largely unresolved. The recent advances in applying single-cell genomics technologies to simultaneously measure the clonality, surface phenotype and transcriptomic signature of pathogen- specific immune cells have significantly improved understanding of these questions. Single-cell multi-omics permits the accurate identification of clonally expanded populations, their differentiation trajectories, the level of immune receptor repertoire diversity involved in the response and the phenotypic and molecular heterogeneity. This thesis aims to develop a bioinformatic workflow utilising single-cell multi-omics data to explore, quantify and predict the clonal and transcriptomic signatures of the human T-cell response during and following viral infection. In the first aim, a web application, VDJView, was developed to facilitate the simultaneous analysis and visualisation of clonal, transcriptomic and clinical metadata of T and B cell multi-omics data. The application permits non-bioinformaticians to perform quality control and common analyses of single-cell genomics data integrated with other metadata, thus permitting the identification of biologically and clinically relevant parameters. The second aim pertains to analysing the functional, molecular and immune receptor profiles of CD8+ T cells in the acute phase of primary hepatitis C virus (HCV) infection. This analysis identified a novel population of progenitors of exhausted T cells, and lineage tracing revealed distinct trajectories with multiple fates and evolutionary plasticity. Furthermore, it was observed that high-magnitude IFN-γ CD8+ T-cell response is associated with the increased probability of viral escape and chronic infection. Finally, in the third aim, a novel analysis is presented based on the topological characteristics of a network generated on pathogen-specific, paired-chain, CD8+ TCRs. This analysis revealed how some cross-reactivity between TCRs can be explained via the sequence similarity between TCRs and that this property is not uniformly distributed across all pathogen-specific TCR repertoires. Strong correlations between the topological properties of the network and the biological properties of the TCR sequences were identified and highlighted. The suite of workflows and methods presented in this thesis are designed to be adaptable to various T and B cell multi-omic datasets. The associated analyses contribute to understanding the role of T and B cells in the adaptive immune response to viral-infection and cancer

    Recombinant spidroins from infinite circRNA translation

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    Spidroins are a diverse family of peptides and the main components of spider silk. They can be used to produce sustainable, lightweight and durable materials for a large variety of medical and engineering applications. Spiders’ territorial behaviour and cannibalism precludes farming them for silk. Recombinant protein synthesis is the most promising way of producing these peptides. However, many approaches have been unsuccessful in obtaining large titres of recombinant spidroins or ones of sufficient molecular weight. The work described here is focused on expressing high molecular weight spidroins from short circular RNA molecules. Mammalian host cells were transfected with designed circular-RNA-producing plasmid vectors. A backsplicing approach was implemented to successfully circularise RNA in a variety of mammalian cell types. This approach could not express any recombinant spidroins based on a variety of qualitative protein assays. Further experiments investigated the reasons behind this. Additionally, due to the diversity of spidroins in a large number of spider lineages, there are potentially many spidroin sequences left to be discovered. A bioinformatic pipeline was developed that accepts transcriptome datasets from RNA sequencing and uses tandem repeat detection and profile HMM annotation to identify novel sequences. This pipeline was specifically designed for the identification of repeat domains in expressed sequences. 21 transcriptomes from 17 different species, encompassing a wide selection of basal and derived spider lineages, were investigated using this pipeline. Six previously undescribed spidroin sequences were discovered. This pipeline was additionally tested in the context of the suckerin protein family. These proteins have recently been investigated for their potential properties in medicine and engineering including adhesion in wet environments. The computational pipeline was able to double the number of suckerins known to date. Further phylogenetic analysis was implemented to expand on the knowledge of suckerins. This pipeline enables the identification of transcripts that may have been overlooked by more mainstream analysis methods such as pairwise homology searches. The spidroins and suckerins discovered by this pipeline may contribute to the large repertoire of potentially useful properties characteristic of this diverse peptide family

    2023-2024 Catalog

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    The 2023-2024 Governors State University Undergraduate and Graduate Catalog is a comprehensive listing of current information regarding:Degree RequirementsCourse OfferingsUndergraduate and Graduate Rules and Regulation

    Second-Person Surveillance: Politics of User Implication in Digital Documentaries

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    This dissertation analyzes digital documentaries that utilize second-person address and roleplay to make users feel implicated in contemporary refugee crises, mass incarceration in the U.S., and state and corporate surveillances. Digital documentaries are seemingly more interactive and participatory than linear film and video documentary as they are comprised of a variety of auditory, visual, and written media, utilize networked technologies, and turn the documentary audience into a documentary user. I draw on scholarship from documentary, game, new media, and surveillance studies to analyze how second-person address in digital documentaries is configured through user positioning and direct address within the works themselves, in how organizations and creators frame their productions, and in how users and players respond in reviews, discussion forums, and Let’s Plays. I build on Michael Rothberg’s theorization of the implicated subject to explore how these digital documentaries bring the user into complicated relationality with national and international crises. Visually and experientially implying that users bear responsibility to the subjects and subject matter, these works can, on the one hand, replicate modes of liberal empathy for suffering, distant “others” and, on the other, simulate one’s own surveillant modes of observation or behavior to mirror it back to users and open up one’s offline thoughts and actions as a site of critique. This dissertation charts how second-person address shapes and limits the political potentialities of documentary projects and connects them to a lineage of direct address from educational and propaganda films, museum exhibits, and serious games. By centralizing the user’s individual experience, the interventions that second-person digital documentaries can make into social discourse change from public, institution-based education to more privatized forms of sentimental education geared toward personal edification and self-realization. Unless tied to larger initiatives or movements, I argue that digital documentaries reaffirm a neoliberal politics of individual self-regulation and governance instead of public education or collective, social intervention. Chapter one focuses on 360-degree virtual reality (VR) documentaries that utilize the feeling of presence to position users as if among refugees and as witnesses to refugee experiences in camps outside of Europe and various dwellings in European cities. My analysis of Clouds Over Sidra (Gabo Arora and Chris Milk 2015) and The Displaced (Imraan Ismail and Ben C. Solomon 2015) shows how these VR documentaries utilize observational realism to make believable and immersive their representations of already empathetic refugees. The empathetic refugee is often young, vulnerable, depoliticized and dehistoricized and is a well-known trope in other forms of humanitarian media that continues into VR documentaries. Forced to Flee (Zahra Rasool 2017), I am Rohingya (Zahra Rasool 2017), So Leben Flüchtlinge in Berlin (Berliner Morgenpost 2017), and Limbo: A Virtual Experience of Waiting for Asylum (Shehani Fernando 2017) disrupt easy immersions into realistic-looking VR experiences of stereotyped representations and user identifications and, instead, can reflect back the user’s political inaction and surveillant modes of looking. Chapter two analyzes web- and social media messenger-based documentaries that position users as outsiders to U.S. mass incarceration. Users are noir-style co-investigators into the crime of the prison-industrial complex in Fremont County, Colorado in Prison Valley: The Prison Industry (David Dufresne and Philippe Brault 2009) and co-riders on a bus transporting prison inmates’ loved ones for visitations to correctional facilities in Upstate New York in A Temporary Contact (Nirit Peled and Sara Kolster 2017). Both projects construct an experience of carceral constraint for users to reinscribe seeming “outside” places, people, and experiences as within the continuation of the racialized and classed politics of state control through mass incarceration. These projects utilize interfaces that create a tension between replicating an exploitative hierarchy between non-incarcerated users and those subject to mass incarceration while also de-immersing users in these experiences to mirror back the user’s supposed distance from this mode of state regulation. Chapter three investigates a type of digital game I term dataveillance simulation games, which position users as surveillance agents in ambiguously dystopian nation-states and force users to use their own critical thinking and judgment to construct the criminality of state-sanctioned surveillance targets. Project Perfect Citizen (Bad Cop Studios 2016), Orwell: Keeping an Eye on You (Osmotic Studios 2016), and Papers, Please (Lucas Pope 2013) all create a dual empathy: players empathize with bureaucratic surveillance agents while empathizing with surveillance targets whose emails, text messages, documents, and social media profiles reveal them to be “normal” people. I argue that while these games show criminality to be a construct, they also utilize a racialized fear of the loss of one’s individual privacy to make players feel like they too could be surveillance targets. Chapter four examines personalized digital documentaries that turn users and their data into the subject matter. Do Not Track (Brett Gaylor 2015), A Week with Wanda (Joe Derry Hall 2019), Stealing Ur Feelings (Noah Levenson 2019), Alfred Premium (Joël Ronez, Pierre Corbinais, and Émilie F. Grenier 2019), How They Watch You (Nick Briz 2021), and Fairly Intelligent™ (A.M. Darke 2021) track, monitor, and confront users with their own online behavior to reflect back a corporate surveillance that collects, analyzes, and exploits user data for profit. These digital documentaries utilize emotional fear- and humor-based appeals to persuade users that these technologies are controlling them, shaping their desires and needs, and dehumanizing them through algorithmic surveillance

    Optical Remote Sensing of Oil Spills by using Machine Learning Methods in the Persian Gulf: A Multi-Class Approach

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    Marine oil spills are harmful for the environment and costly for society. Coastal areas are particularly vulnerable since they provide habitats for organisms, animals and marine ecosystems. This thesis studied machine learning methods to classify thick oil in a multi-class case, using remotely sensed multi-spectral data in the Persian Gulf. The study area covers a large area between United Arab Emirates (UAE) and Iran. The dataset is extracted from 10 Sentinel-2 tiles on six spectral bands between 492 nm to 2202 nm. These images were annotated for four classes, namely thick oil, thin oil, ocean water and turbid water by using the Bonn Agreement to analyse true color composite images. A variety of machine learning methods were trained and evaluated using this dataset. Then a robustness evaluation was done by using selected machine learning methods on an independent dataset. Initially multiple machine learning methods were included; three decision trees, six K-Nearest Neighbor (KNN) models, two Artificial Neural Network (ANN) models, two Naive bayes models, and two discriminant models. Two KNN models and two ANN models were then picked for further evaluation. The results show that the fine KNN approach with two nearest neighbors had the best performance based on the computed statistical measures. However, the robustness evaluation showed that the tri-layered NN performed better. This thesis has shown that supervised machine learning with a multi-class approach can be used for oil spill monitoring using multi-spectral remote sensing data in the Persian Gulf

    Dissecting regional heterogeneity and modeling transcriptional cascades in brain organoids

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    Over the past decade, there has been a rapid expansion in the development and utilization of brain organoid models, enabling three-dimensional in vivo-like views of fundamental neurodevelopmental features of corticogenesis in health and disease. Nonetheless, the methods used for generating cortical organoid fates exhibit widespread heterogeneity across different cell lines. Here, we show that a combination of dual SMAD and WNT inhibition (Triple-i protocol) establishes a robust cortical identity in brain organoids, while other widely used derivation protocols are inconsistent with respect to regional specification. In order to measure this heterogeneity, we employ single-cell RNA-sequencing (scRNA-Seq), enabling the sampling of the gene expression profiles of thousands of cells in an individual sample. However, in order to draw meaningful conclusions from scRNA-Seq data, technical artifacts must be identified and removed. In this thesis, we present a method to detect one such artifact, empty droplets that do not contain a cell and consist mainly of free-floating mRNA in the sample. Furthermore, from their expression profiles, cells can be ordered along a developmental trajectory which recapitulates the progression of cells as they differentiate. Based on this ordering, we model gene expression using a Bayesian inference approach in order to measure transcriptional dynamics within differentiating cells. This enables the ordering of genes along transcriptional cascades, statistical testing for differences in gene expression changes, and measuring potential regulatory gene interactions. We apply this approach to differentiating cortical neural stem cells into cortical neurons via an intermediate progenitor cell type in brain organoids to provide a detailed characterization of the endogenous molecular processes underlying neurogenesis.Im letzten Jahrzent hat die Entwicklung und Nutzung von Organoidmodellen des Gehirns stark zugenommen. Diese Modelle erlauben dreidimensionale, in-vivo ähnliche Einblicke in fundamentale Aspekte der neurologischen Entwicklung des Hirnkortex in Gesundheit und Krankheit. Jedoch weisen die Methoden, um die Entwicklung kortikaler Organoide zu verfolgen, starke Heterogenität zwischen verschiedenen Zelllinien auf. Hier weisen wir nach, dass eine Kombination dualer SMAD und WNT Hemmung (Triple-i Protokoll) eine konstante kortikale Zuordnung in Hirnorganoiden erzeugt, während andere, weit verbreitete und genutzte Protokolle in Bezug auf kortikale Spezifizierung keine konstanten Ergebnisse liefern. Um die Heterogenität zu messen, haben wir Einzelzell-RNA Sequenzierung (scRNA-Seq) benutzt, wodurch die Erfassung der Genexpression von Tausenden von Zellen in einer Probe möglich ist. Um jedoch sinnvolle Schlüsse aus diesen scRNA-Seq Daten zu ziehen, müssen technische Artifakte identifiziert und aus den Daten entfernt werden. In dieser Dissertation stellen wir eine Methode vor, um eines solcher Artifakte zu erkennen: leere Tröpfchen (ohne Zellen), die hauptsächlich aus freischwebender mRNA in der Probe bestehen. Weiterhin können Zellen anhand ihrer Genexpressionsprofile entlang einer Entwicklungsschiene angeordnet werden, die die Entwicklung der Zellen während ihrer Differenzierung rekapituliert. Auf der Grundlage dieser Entwicklungsreihenfolge modellieren wir die Genexpression mit einem Bayes’schen Inferenzansatz, um die Dynamik der Transkription in sich differenzierenden Zellen zu messen. Dies ermöglicht das Anordnen von Genen entlang einer Transkriptionskaskade, sowie statistische Untersuchungen in Hinblick auf Unterschiede in der Veränderung von Genexpression, und das Messen des Einflusses möglicher Regulationsgene. Wir wenden diese Methode an, um kortikale neuronale Stammzellen zu untersuchen, die sich über einen intermediären Vorläuferzelltyp in kortikale Neuronen in Hirnorganoiden differenzieren, und um eine detaillierte Charakterisierung der molekularen Prozesse zu liefern, die der Neurogenese zugrunde liegen
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