8,758 research outputs found

    Transcriptomic analysis of canine B and T cell lymphoma

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    Gene expression profiling in human lymphoma has identified a number of genes and pathways of prognostic value that could also be used as targets for therapy. The aim of the present study was to compare gene expression in canine B-cell lymphoma, T-cell lymphoma and control lymph node samples using RNA-Seq to identify differentially expressed pathways which may have a role in pathogenesis and could be used as possible treatment targets. RNA was extracted from lymph node tissue collected from six dogs with B-cell lymphoma, five dogs with T-cell lymphoma, one dog with B and T-cell lymphoma and eight controls. The RNA was sequenced using an Illumina NextSeq500 platform, reads were aligned to the current canine genome and gene expression profiles were determined for each group. Pathway analysis was also performed to identify networks enriched for differentially expressed genes together with activated and repressed molecular pathways. After stand-alone analysis of the dataset, it was combined with a previous dataset representing eighteen dogs with B-cell lymphoma and five dogs with T-cell lymphoma in order to increase the power of the analysis. Principal component analysis revealed that the three groups, i.e. B-cell lymphoma, T-cell lymphoma and controls, formed separate clusters with distinct expression signatures. Network analysis highlighted different pathways overrepresented in each type of lymphoma. In B-cell lymphoma tissue, the NF-ĸB pathway, cell cycle control and DNA repair pathways were over-represented with overexpression of ribosomal and minichromosome maintenance proteins also noted. In contrast for T-cell lymphomas, mRNA synthesis, FGFR and RUNX pathways were highlighted together with those relating to innate immunity. This project identified several aberrant pathways in B-cell and T-cell lymphoma that have not previously been reported and which can be considered candidate targets for developing novel treatment, thereby informing future studies

    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

    Advancements and future prospects of adeno-associated virus-mediated gene therapy for sensorineural hearing loss

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    Sensorineural hearing loss (SNHL), a highly prevalent sensory impairment, results from a multifaceted interaction of genetic and environmental factors. As we continually gain insights into the molecular basis of auditory development and the growing compendium of deafness genes identified, research on gene therapy for SNHL has significantly deepened. Adeno-associated virus (AAV), considered a relatively secure vector for gene therapy in clinical trials, can deliver various transgenes based on gene therapy strategies such as gene replacement, gene silencing, gene editing, or gene addition to alleviate diverse types of SNHL. This review delved into the preclinical advances in AAV-based gene therapy for SNHL, spanning hereditary and acquired types. Particular focus is placed on the dual-AAV construction method and its application, the vector delivery route of mouse inner ear models (local, systemic, fetal, and cerebrospinal fluid administration), and the significant considerations in transforming from AAV-based animal model inner ear gene therapy to clinical implementation

    Genetic and epigenetic characterization of cell-free DNA:In patients with solid tumors

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    Investigating the effects of palmitoylation on the dopamine 1 receptor (D1)

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    The dopamine D1 receptor (D1) is a G protein-coupled receptor (GPCR) which regulates various key brain functions like attention, movement, reward, and memory. Understanding D1 signalling may open the horizon for novel treatments for neurological disorders. Upon agonist activation, the heterotrimeric G proteins Gαs activate adenylyl cyclase to increase cAMP/PKA signalling. D1 also engages β-arrestin proteins leading to β-arrestin dependent signalling. The D1 has two palmitoylation sites on cysteines 347&351 in its C-tail domain. However, the distinct roles and implications of palmitoylation on the D1 signalling, trafficking and β-arrestins recruitment are still largely unexplored. A palmitoylation D1 mutant was generated and luminescent based techniques such as BRET and split-Nanoluc complementation assay were employed, to delineate D1 palmitoylation effects on its pharmacology and signalling. The D1 agonists induced 50% less cAMP production in the mutant compared to wildtype (WT) and WT showed a more efficient dissociation of its Gαs. Moreover, the mutant receptor failed to recruit β-arrestin1&2, induced less ERK1/2 activation and internalises in an agonist-independent process while showing an altered intracellular Golgi trafficking. Also, in β-arrestin 1&2 KO HEK 293 cells similar cAMP production levels were reported for D1 WT and palmitoylation mutant. β-arrestin 1&2 KO blocked agonist-induced WT D1 plasma membrane trafficking, indicating that these β-arrestins are driving the differences between WT and the palmitoylation mutant D1. Taken together, our studies indicate that Gαs is the main transducer for D1 cAMP and ERK1/2 signalling and that palmitoylation is essential for its β-arrestin 1&2 interactions and modulating D1 signalling cascades in a drug-dependant process

    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

    Netrin-1 isoforms and macrophage phenotype: role in the pathophysiology of atherosclerosis

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    Atherosclerosis is a chronic inflammatory disease and is the main cause of ischaemic heart disease, stroke, and other cardiovascular diseases. The development of atherosclerotic plaques incidence has been increasing significantly during the last few decades and is due to different factors, such as the increased consumption of high fat food, smoking and increased incidence of diabetes mellitus. This is currently a major burden on health systems, which require new therapeutic targets to address this challenge. The involvement of netrin-1 in inflammation and atherosclerosis has been studied during the last two decades. Different groups have established that netrin-1 plays a prominent role in these contexts and has different effects depending on where it is produced, and which cells are targeted by it. While endothelial-derived netrin-1 secreted into the circulation gives rise to a protective effect against atherosclerosis, macrophage-derived netrin-1 within the plaque has a pro-atherogenic effect, promoting the trapping and survivability of foam cells. More recently, a truncated isoform of netrin-1 was found in the nuclei of different types of cancer cells. This form of netrin-1 has also been studied in endothelial cells, but so far, no relationship between the expression of truncated netrin-1 and macrophages has been established. The link between cytokine stimulation, macrophage phenotype and netrin-1 isoforms has until now been unclear. The work described in this thesis looked at the expression and function of netrin-1 on monocytes and different macrophage phenotypes. Gene expression analysis revealed that macrophages express both full-length and truncated netrin-1. Classical activated is the macrophage phenotype that presents higher expression of netrin-1, and the expression of both isoforms is at least partially dependent on NF-κB activation pathway. Netrin-1 inhibits monocyte migration by inhibiting chemokines’ chemoattractant effect. CCL2 and netrin-1 individually showed an attractant effect towards THP-1-derived macrophages, but the signals were inhibited when the two were combined. The apoptotic agent selected to study netrin-1 anti-apoptotic effect was not UNC5b-dependent and, therefore, limited our ability to acquire relevant data regarding this process. Administration of exogenous netrin-1 to mice, increasing its systemic levels, showed an acute and chronic protective effect against inflammation. Mice treated with netrin-1 showed less macrophages within the tissue after the induction of local inflammation. Furthermore, increasing the netrin-1 systemic levels of mice prevented the enlargement of the aortic sinus and development of plaque after feeding them 60% HFD over a 6-week period. This thesis provides relevant insights into the role of macrophage phenotypes in the expression of netrin-1 isoforms within atherosclerotic plaques, and how netrin-1 affects these immune cells. The findings highlight the significance of netrin-1 as a potential therapeutic target for treating not only cardiovascular disease but also other inflammatory conditions. Therefore, this research sheds light on the promising future of netrin-1 as a treatment option for a range of diseases

    Clinical, immunological and genetic features of histiocytic disorders

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