1,780 research outputs found

    Exploring missing heritability in neurodevelopmental disorders:Learning from regulatory elements

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    In this thesis, I aimed to solve part of the missing heritability in neurodevelopmental disorders, using computational approaches. Next to the investigations of a novel epilepsy syndrome and investigations aiming to elucidate the regulation of the gene involved, I investigated and prioritized genomic sequences that have implications in gene regulation during the developmental stages of human brain, with the goal to create an atlas of high confidence non-coding regulatory elements that future studies can assess for genetic variants in genetically unexplained individuals suffering from neurodevelopmental disorders that are of suspected genetic origin

    Advanced sequencing technologies applied to human cytomegalovirus

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    The betaherpesvirus human cytomegalovirus (HCMV) is a ubiquitous viral pathogen. It is the most common cause of congenital infection in infants and of opportunistic infections in immunocompromised patients worldwide. The large double-stranded DNA genome of HCMV (236 kb) contains several genes that exhibit a high degree of variation among strains within an otherwise highly conserved sequence. These hypervariable genes encode immune escape, tropism or regulatory factors that may affect virulence. Variation arising from these genes and from an evolutionary history of recombination between strains has been hypothesised to be linked to disease severity. To investigate this, the HCMV genome has been scrutinised in detail over the years using a variety of molecular techniques, most looking only at one or a few of these genes at a time. The advent of high-throughput sequencing (HTS) technology 20 years ago then started to enable more in-depth whole-genome analyses. My study extends this field by using both HTS and the more recently developed long-read nanopore technology to determine HCMV genome sequences directly from clinical samples. Firstly, I used an Illumina HTS pipeline to sequence HCMV strains directly from formalin-fixed, paraffin-embedded (FFPE) tissues. FFPE samples are a valuable repository for the study of relatively rare diseases, such as congenital HCMV (cCMV). However, formalin fixation induces DNA fragmentation and cross-linking, making this a challenging sample type for DNA sequencing. I successfully sequenced five whole HCMV genomes from FFPE tissues. Next, I developed a pipeline utilising the single-molecule, long-read sequencer from Oxford Nanopore Technologies (ONT) to sequence HCMV initially from high-titre cellcultured laboratory strains and then from clinical samples with high HCMV loads. Finally, I utilised a direct RNA sequencing protocol with the ONT sequencer to characterise novel HCMV transcripts produced during infection in cell culture, demonstrating the existence of transcript isoforms with multiple splice sites. Overall, my findings demonstrate how advanced sequencing technologies can be used to characterise the genome and transcriptome of a large DNA virus, and will facilitate future studies on HCMV prognostic factors, novel antiviral targets and vaccine development

    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/

    Analysis and monitoring of single HaCaT cells using volumetric Raman mapping and machine learning

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    No explorer reached a pole without a map, no chef served a meal without tasting, and no surgeon implants untested devices. Higher accuracy maps, more sensitive taste buds, and more rigorous tests increase confidence in positive outcomes. Biomedical manufacturing necessitates rigour, whether developing drugs or creating bioengineered tissues [1]–[4]. By designing a dynamic environment that supports mammalian cells during experiments within a Raman spectroscope, this project provides a platform that more closely replicates in vivo conditions. The platform also adds the opportunity to automate the adaptation of the cell culture environment, alongside spectral monitoring of cells with machine learning and three-dimensional Raman mapping, called volumetric Raman mapping (VRM). Previous research highlighted key areas for refinement, like a structured approach for shading Raman maps [5], [6], and the collection of VRM [7]. Refining VRM shading and collection was the initial focus, k-means directed shading for vibrational spectroscopy map shading was developed in Chapter 3 and exploration of depth distortion and VRM calibration (Chapter 4). “Cage” scaffolds, designed using the findings from Chapter 4 were then utilised to influence cell behaviour by varying the number of cage beams to change the scaffold porosity. Altering the porosity facilitated spectroscopy investigation into previously observed changes in cell biology alteration in response to porous scaffolds [8]. VRM visualised changed single human keratinocyte (HaCaT) cell morphology, providing a complementary technique for machine learning classification. Increased technical rigour justified progression onto in-situ flow chamber for Raman spectroscopy development in Chapter 6, using a Psoriasis (dithranol-HaCaT) model on unfixed cells. K-means-directed shading and principal component analysis (PCA) revealed HaCaT cell adaptations aligning with previous publications [5] and earlier thesis sections. The k-means-directed Raman maps and PCA score plots verified the drug-supplying capacity of the flow chamber, justifying future investigation into VRM and machine learning for monitoring single cells within the flow chamber

    The use of biofluid markers to evaluate the consequences of sport-related subconcussive head impact exposure : a scoping review

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    This review was supported by the University of Stirling (no grant number applies). L.W. also received support as part of Framework 7 programme of the European Union (CENTER-TBI, Grant number: 602150–2). S.M. received research support from the Italian Ministry of Health (GR-2013–02354960).Background Amidst growing concern about the safety of sport-related repetitive subconcussive head impacts (RSHI), biofluid markers may provide sensitive, informative, and practical assessment of the effects of RSHI exposure. Objective This scoping review aimed to systematically examine the extent, nature, and quality of available evidence from studies investigating the effects of RSHI on biofluid markers, to identify gaps and to formulate guidelines to inform future research. Methods PRISMA extension for Scoping Reviews guidelines were adhered to. The protocol was pre-registered through publication. MEDLINE, Scopus, SPORTDiscus, CINAHL, PsycINFO, Cochrane Library, OpenGrey, and two clinical trial registries were searched (until March 30, 2022) using descriptors for subconcussive head impacts, biomarkers, and contact sports. Included studies were assessed for risk of bias and quality. Results Seventy-nine research publications were included in the review. Forty-nine studies assessed the acute effects, 23 semi-acute and 26 long-term effects of RSHI exposure. The most studied sports were American football, boxing, and soccer, and the most investigated markers were (in descending order): S100 calcium-binding protein beta (S100B), tau, neurofilament light (NfL), glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), brain-derived neurotrophic factor (BDNF), phosphorylated tau (p-tau), ubiquitin C-terminal hydrolase L1 (UCH-L1), and hormones. High or moderate bias was found in most studies, and marker-specific conclusions were subject to heterogeneous and limited evidence. Although the evidence is weak, some biofluid markers—such as NfL—appeared to show promise. More markedly, S100B was found to be problematic when evaluating the effects of RSHI in sport. Conclusion Considering the limitations of the evidence base revealed by this first review dedicated to systematically scoping the evidence of biofluid marker levels following RSHI exposure, the field is evidently still in its infancy. As a result, any recommendation and application is premature. Although some markers show promise for the assessment of brain health following RSHI exposure, future large standardized and better-controlled studies are needed to determine biofluid markers’ utility.Publisher PDFPeer reviewe

    Exploring missing heritability in neurodevelopmental disorders:Learning from regulatory elements

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    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

    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-Îł 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

    Investigation of the metabolism of rare nucleotides in plants

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    Nucleotides are metabolites involved in primary metabolism, and specialized metabolism and have a regulatory role in various biochemical reactions in all forms of life. While in other organisms, the nucleotide metabolome was characterized extensively, comparatively little is known about the cellular concentrations of nucleotides in plants. The aim of this dissertation was to investigate the nucleotide metabolome and enzymes influencing the composition and quantities of nucleotides in plants. For this purpose, a method for the analysis of nucleotides and nucleosides in plants and algae was developed (Chapter 2.1), which comprises efficient quenching of enzymatic activity, liquid-liquid extraction and solid phase extraction employing a weak-anionexchange resin. This method allowed the analysis of the nucleotide metabolome of plants in great depth including the quantification of low abundant deoxyribonucleotides and deoxyribonucleosides. The details of the method were summarized in an article, serving as a laboratory protocol (Chapter 2.2). Furthermore, we contributed a review article (Chapter 2.3) that summarizes the literature about nucleotide analysis and recent technological advances with a focus on plants and factors influencing and hindering the analysis of nucleotides in plants, i.e., a complex metabolic matrix, highly stable phosphatases and physicochemical properties of nucleotides. To analyze the sub-cellular concentrations of metabolites, a protocol for the rapid isolation of highly pure mitochondria utilizing affinity chromatography was developed (Chapter 2.4). The method for the purification of nucleotides furthermore contributed to the comprehensive analysis of the nucleotide metabolome in germinating seeds and in establishing seedlings of A. thaliana, with a focus on genes involved in the synthesis of thymidilates (Chapter 2.5) and the characterization of a novel enzyme of purine nucleotide degradation, the XANTHOSINE MONOPHOSPHATE PHOSPHATASE (Chapter 2.6). Protein homology analysis comparing A. thaliana, S. cerevisiae, and H. sapiens led to the identification and characterization of an enzyme involved in the metabolite damage repair system of plants, the INOSINE TRIPHOSPHATE PYROPHOSPHATASE (Chapter 2.7). It was shown that this enzyme dephosphorylates deaminated purine nucleotide triphosphates and thus prevents their incorporation into nucleic acids. Lossof-function mutants senesce early and have a constitutively increased content of salicylic acid. Also, the source of deaminated purine nucleotides in plants was investigated and it was shown that abiotic factors contribute to nucleotide damage.Nukleotide sind Metaboliten, die am Primärstoffwechsel und an spezialisierten Stoffwechselvorgängen beteiligt sind und eine regulierende Rolle bei verschiedenen biochemischen Reaktionen in allen Lebensformen spielen. Während bei anderen Organismen das Nukleotidmetabolom umfassend charakterisiert wurde, ist in Pflanzen vergleichsweise wenig über die zellulären Konzentrationen von Nukleotiden bekannt. Ziel dieser Dissertation war es, das Nukleotidmetabolom und die Enzyme zu untersuchen, die die Zusammensetzung und Menge der Nukleotide in Pflanzen beeinflussen. Zu diesem Zweck wurde eine Methode zur Analyse von Nukleotiden und Nukleosiden in Pflanzen und Algen entwickelt (Kapitel 2.1), die ein effizientes Stoppen enzymatischer Aktivität, eine Flüssig-Flüssig-Extraktion und eine Festphasenextraktion unter Verwendung eines schwachen Ionenaustauschers umfasst. Mit dieser Methode konnte das Nukleotidmetabolom von Pflanzen eingehend analysiert werden, einschließlich der Quantifizierung von Desoxyribonukleotiden und Desoxyribonukleosiden mit geringer Abundanz. Die Einzelheiten der Methode wurden in einem Artikel zusammengefasst, der als Laborprotokoll dient (Kapitel 2.2). Darüber hinaus wurde ein Übersichtsartikel (Kapitel 2.3) verfasst, der die Literatur über die Analyse von Nukleotiden und die jüngsten technologischen Fortschritte zusammenfasst. Der Schwerpunkt lag hierbei auf Pflanzen und Faktoren, die die Analyse von Nukleotiden in Pflanzen beeinflussen oder behindern, d. h. eine komplexe Matrix, hochstabile Phosphatasen und physikalisch-chemische Eigenschaften von Nukleotiden. Um die subzellulären Konzentrationen von Metaboliten zu analysieren, wurde ein Protokoll für die schnelle Isolierung hochreiner Mitochondrien unter Verwendung einer Affinitätschromatographie entwickelt (Kapitel 2.4). Die Methode zur Analyse von Nukleotiden trug außerdem zu einer umfassenden Analyse des Nukleotidmetaboloms in keimenden Samen und in sich etablierenden Keimlingen von A. thaliana bei, wobei der Schwerpunkt auf Genen lag, die an der Synthese von Thymidilaten beteiligt sind (Kapitel 2.5), sowie zu der Charakterisierung eines neuen Enzyms des Purinnukleotidabbaus, der XANTHOSINE MONOPHOSPHATE PHOSPHATASE (Kapitel 2.6). Eine Proteinhomologieanalyse, die A. thaliana, S. cerevisiae und H. sapiens miteinander verglich führte zur Identifizierung und Charakterisierung eines Enzyms, das an der Reparatur von geschädigten Metaboliten in Pflanzen beteiligt ist, der INOSINE TRIPHOSPHATE PYROPHOSPHATASE (Kapitel 2.7). Es konnte gezeigt werden, dass dieses Enzym desaminierte Purinnukleotidtriphosphate dephosphoryliert und so deren Einbau in Nukleinsäuren verhindert. Funktionsverlustmutanten altern früh und weisen einen konstitutiv erhöhten Gehalt an Salicylsäure auf. Außerdem wurde die Quelle der desaminierten Purinnukleotide in Pflanzen untersucht, und es wurde gezeigt, dass abiotische Faktoren zur Nukleotidschädigung beitragen
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