2,533 research outputs found

    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

    Identifying alterations in adipose tissue-derived islet GPCR peptide ligand mRNAs in obesity: implications for islet function

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    In addition to acting as an energy reservoir, white adipose tissue is a vital endocrine organ involved in the modulation of cellular function and the maintenance of metabolic homeostasis through the synthesis and secretion of peptides, known as adipokines. It is known that some of these secretory peptides play important regulatory roles in glycaemic control by acting directly on islet β-cells or on insulin-sensitive tissues. Excess adiposity causes alterations in the circulating levels of some adipokines which, depending on their mode of action, can have pro-inflammatory, pro-diabetic or anti-inflammatory, anti-diabetic properties. Some adipokines that are known to act at β-cells have actions that are transduced by binding to G protein- coupled receptors (GPCRs). This large family of receptors represents ~35% of all current drug targets for the treatment of a wide range of diseases, including type 2 diabetes (T2D). Islets express ~300 GPCRs, yet only one islet GPCR is currently directly targeted for T2D treatment. This deficit represents a therapeutic gap that could be filled by the identification of adipose tissue-derived islet GPCR peptide ligands that increase insulin secretion and overall β-cell function. Thus, by defining their mechanisms of action, there is potential for the development of new pharmacotherapies for T2D. Therefore, this thesis describes experiments which aimed to compare the expression profiles of adipose tissue-derived islet GPCR peptide ligand mRNAs under lean and obese conditions, and to characterise the functional effects of a selected candidate of interest on islet cells. Visceral fat depots were retrieved from high-fat diet-induced and genetically obese mouse models, and from human participants. Fat pads were either processed as whole tissue, or mature adipocyte cells were separated from the stromal vascular fraction (SVF) which contains several other cell populations, including preadipocytes and macrophages. The expression levels of 155 islet GPCR peptide ligand mRNAs in whole adipose tissue or in isolated mature adipocytes were quantified using optimised RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocols. Comparisons between lean and obese states in mice models and humans revealed significant modifications in the expression levels of several adipokine mRNAs. As expected, mRNAs encoding the positive control genes, Lep and AdipoQ were quantifiable, with the expression of Lep mRNA increasing and that of AdipoQ mRNA decreasing in obesity. Expression of Ccl4 mRNA, encoding chemokine (C-C motif) ligand 4, was significantly upregulated in whole adipose tissue across all models of obesity compared to their lean counterparts. This coincided with elevated circulating Ccl4 peptide levels. This increase was not replicated in isolated mature adipocytes, indicating that the source of upregulated Ccl4 expression in obesity was the SVF of adipose tissue. Based on this significant increase in Ccl4 mRNA expression within visceral fat and its undetermined effects on β-cell function, Ccl4 was selected for further investigation in MIN6 β-cells and mouse islets. PRESTO-Tango β-arrestin reporter assays were performed to determine which GPCRs were activated by exogenous Ccl4. Experiments using HTLA cells expressing a protease-tagged β- arrestin and transfected with GPCR plasmids of interest indicated that 100ng/mL Ccl4 significantly activated Cxcr1 and Cxcr5, but it was not an agonist at the previously identified Ccl4-target GPCRs Ccr1, Ccr2, Ccr5, Ccr9 and Ackr2. RNA extraction and RT-qPCR experiments using MIN6 β-cells and primary islets from lean mice revealed the expression of Cxcr5 mRNA in mouse islets, but it was absent in MIN6 β-cells. The remaining putative Ccl4 receptors (Ccr1, Ccr2, Ccr5, Ccr9, Cxcr1 and Ackr2) were either absent or present at trace levels in mouse islets and MIN6 β-cells. Recombinant mouse Ccl4 protein was used for functional experiments at concentrations of 5, 10, 50 and 100ng/mL, based on previous reports of biological activities at these concentrations. Trypan blue exclusion testing was initially performed to assess the effect of exogenous Ccl4 on MIN6 β-cell viability and these experiments indicated that all concentrations (5-100ng/mL) were well-tolerated. Since β-cells have a low basal rate of apoptosis, cell death was induced by exposure to the saturated free fatty acid, palmitate, or by a cocktail of pro-inflammatory cytokines (interleukin-1β, tumour necrosis factor-α and interferon-γ). In MIN6 β-cells, Ccl4 demonstrated concentration-dependent protective effects against palmitate-induced and cytokine-induced apoptosis. Conversely, while palmitate and cytokines also increased apoptosis of mouse islets, Ccl4 did not protect islets from either inducer. Quantification of bromodeoxyuridine (BrdU) incorporation into β-cell DNA indicated that Ccl4 caused a concentration-dependent reduction in proliferation of MIN6 β-cells in response to 10% fetal bovine serum (FBS). In contrast, immunohistochemical quantification of Ki67-positive mouse islet β-cells showed no differences in β-cell proliferation between control- and Ccl4-treated islets. Whilst the number of β-cells and δ-cells were unaffected, α- cells were significantly depleted by Ccl4 treatment. Exogenous Ccl4 had no effect on nutrient- stimulated insulin secretion from both MIN6 β-cells and primary mouse islets. The 3T3-L1 preadipocyte cell line was used to assess potential Ccl4-mediated paracrine and/or autocrine signalling within adipose tissue. Ccl4 did not alter the mRNA expression of Pparγ, a master regulator of adipocyte differentiation, but did significantly downregulate the mRNA expression of the crucial adipogenic gene, adiponectin. Oil Red O staining and Western blotting were performed to assess lipid accumulation, and insulin and lipolytic signalling, respectively, and these experiments indicated that the observed Ccl4-induced decrease in adiponectin expression failed to correlate with any changes in adipocyte function. In summary, these data demonstrated anti-apoptotic and anti-proliferative actions of the adipokine, Ccl4, on MIN6 β-cells that were not replicated in mouse islets. The absence of any anti-apoptotic, insulin secretory and/or pro-proliferative effects of Ccl4 in islet β-cells suggests that it is unlikely to play a role in regulating β-cell function via crosstalk between adipose tissue and islets. The divergent functional effects highlight that whilst MIN6 cells are a useful primary β-cell surrogate for some studies, primary islets should always be used to confirm physiological relevance. On the other hand, significant α-cell depletion following Ccl4 treatment suggests a cell-specific function within the islets. Furthermore, Ccl4 impaired adiponectin mRNA expression in adipocytes, although, how adipocyte function is affected as a result requires further investigation. Collectively, these data have contributed increased understanding of the role of obesity in modifying the expression of adipose tissue-derived islet GPCR peptide ligands

    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

    Management of socio-economic transformations of business processes: current realities, global challenges, forecast scenarios and development prospects

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    The authors of the scientific monograph have come to the conclusion that мanagement of socio-economic transformations of business processes requires the use of mechanisms to support of entrepreneurship, sectors of the national economy, the financial system, and critical infrastructure. Basic research focuses on assessment the state of social service provision, analysing economic security, implementing innovation and introducing digital technologies. The research results have been implemented in the different models of costing, credit risk and capital management, tax control, use of artificial intelligence and blockchain. The results of the study can be used in the developing of policies, programmes and strategies for economic security, development of the agricultural sector, transformation of industrial policy, implementation of employment policy in decision-making at the level of ministries and agencies that regulate the management of socio-economic and European integration processes. The results can also be used by students and young scientists in the educational process and conducting scientific research on global challenges and creation scenarios for the development of socio-economic processes

    Towards trustworthy computing on untrustworthy hardware

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    Historically, hardware was thought to be inherently secure and trusted due to its obscurity and the isolated nature of its design and manufacturing. In the last two decades, however, hardware trust and security have emerged as pressing issues. Modern day hardware is surrounded by threats manifested mainly in undesired modifications by untrusted parties in its supply chain, unauthorized and pirated selling, injected faults, and system and microarchitectural level attacks. These threats, if realized, are expected to push hardware to abnormal and unexpected behaviour causing real-life damage and significantly undermining our trust in the electronic and computing systems we use in our daily lives and in safety critical applications. A large number of detective and preventive countermeasures have been proposed in literature. It is a fact, however, that our knowledge of potential consequences to real-life threats to hardware trust is lacking given the limited number of real-life reports and the plethora of ways in which hardware trust could be undermined. With this in mind, run-time monitoring of hardware combined with active mitigation of attacks, referred to as trustworthy computing on untrustworthy hardware, is proposed as the last line of defence. This last line of defence allows us to face the issue of live hardware mistrust rather than turning a blind eye to it or being helpless once it occurs. This thesis proposes three different frameworks towards trustworthy computing on untrustworthy hardware. The presented frameworks are adaptable to different applications, independent of the design of the monitored elements, based on autonomous security elements, and are computationally lightweight. The first framework is concerned with explicit violations and breaches of trust at run-time, with an untrustworthy on-chip communication interconnect presented as a potential offender. The framework is based on the guiding principles of component guarding, data tagging, and event verification. The second framework targets hardware elements with inherently variable and unpredictable operational latency and proposes a machine-learning based characterization of these latencies to infer undesired latency extensions or denial of service attacks. The framework is implemented on a DDR3 DRAM after showing its vulnerability to obscured latency extension attacks. The third framework studies the possibility of the deployment of untrustworthy hardware elements in the analog front end, and the consequent integrity issues that might arise at the analog-digital boundary of system on chips. The framework uses machine learning methods and the unique temporal and arithmetic features of signals at this boundary to monitor their integrity and assess their trust level

    Schooling of refugee youth and its effect on their identity = ナンミン セイショウネン ノ ガッコウ キョウイク ト アイデンティティ ケイセイ

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    PDF/A formatsAccess: via World Wide Web東京外国語大学大学院総合国際学研究科博士 (学術) 論文 (2023年1月)Author's thesis (Ph.D)--Tokyo University of Foreign Studies, 2023博甲第341号Bibliography: p. 251-330東京外国語大学 (Tokyo University of Foreign Studies)博士 (学術

    Proteomic Analysis of the Unfolded Protein Response in Melanoma

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    Despite enormous advancements made in the last decade in the treatment of metastatic melanoma, due to drug resistance and drug toxicities, new therapies and treatment strategies are needed. Additionally, there are no prognostic biomarkers for metastatic disease able to predict outcome. Thirty-two potential biomarkers were analysed by selected reaction monitoring (SRM) in 30 stage III melanoma patients. A 14-protein panel was discovered, able to predict patients likely to have poor outcome and therefore potentially benefit from aggressive therapeutic strategies. From the above study, the Unfolded Protein Response (UPR) was revealed to be a major cellular pathway up-regulated in patients with poor outcome. The UPR is a cellular stress response, which is initiated by a build-up of unfolded protein in the endoplasmic reticulum (ER). Increased activation of the UPR is associated with several cancers however, the mechanisms used to promote tumour progression and metastases are not well understood. To characterise this stress response, the UPR was activated in melanoma cell line models. Using quantitative mass spectrometry 64 proteins were identified as differentially abundant with increased UPR activation. Among them, eight UPR-associated proteins were validated by SRM, identifying these proteins as core modulators of the UPR. An in silico analysis of the eight UPR-associated proteins in pan-cancer patient data across 16 solid tumour types revealed the eight UPR-associated proteins were markers of poor survival across cancer types. The combined data demonstrates the UPR is a major contributor to cancer progression. The study contributes to our knowledge of melanoma biology by elucidating the broad impact of the UPR on several cellular pathways and mechanisms that would promote tumour growth and increase the metastatic potential of melanoma. Furthermore, novel UPR drug targets were identified, including cooperative pathways that could be targeted in combinatorial therapie
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