Histone acetylation and inflammatory mediators in inflammatory bowel disease

A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy of the University of LutonDuring cell activation the tightly compacted DNA is made available to DNA-binding proteins allowing the induction of gene transcription. In the resting cell, DNA is packaged into chromatin whose fundamental subunit is the nucleosome, composed of an octamer of four core histones (H) 3, 4, 2A and 2B. During the induction of gene transcription, modification of histones, by acetylation, methylation etc., results in unwinding of the DNA, permitting access of large DNAbinding proteins, such as RNA polymerase II, and subsequent induction of gene transcription. This investigation initially examined the effects of pro-inflammatory stimuli LPS and TNF-a on the production of IL-8 in a macrophage cell line (U937 cells) and in two T-cell lines (Jurkat and HUT-78 cells) as a marker of NF-KB-directed inflammatory gene expression. The ability of dexamethasone (Dex) and triamcinolone acetonide (TA) (synthetic glucocorticoid agonists) to suppress expression of the inflammatory cytokine IL-8 and to regulate histone acetylation was also investigated in these cells. LPS and TNF-a caused an increase in IL-8 expression, which was further enhanced by the histone deacetylases inhibitor trichostatin A (TSA), suggesting a role for histone acetylation in IL-8 production in these cells. Dex and TA, repressed LPS- and TNF-a -induced IL-8 expression in all three cell lines. This effect of both Dex and TA was attenuated by TSA in all cell lines studied, where the effect of TSA was greater in TA stimulated cells. Stimulation of all cell lines with LPS and TNF-a induced acetylation of H4 lysine residues (K5, 8, 12 and 16), the highest elevation of which was for K8 and K12. Also demonstrate is a K5 and K16 specificity of acetylation by glucocorticoids, apparent in all cell lines studied. Dex and, to a greater extent, TA suppressed LPS- and TNFa-induced K8 and K12 acetylation. TSA attenuated the inhibitory effect of the glucocorticoids for all three cell lines. An inCrease in HDAC activity with GCs was observed and ChiP assay showed these events occur on the native IL-8 promoter via histone acetylation. Further studies investigated whether there were any links between histone acetylation and the regulation of apoptosis. It was showed that TSA induced apoptosis in cells previously stimulated with the inducer of oxidative stress hydrogen peroxide (H20 2). Studies into the activation of caspase 3 in LPS- and TNF-a stimulated cells revealed that the combinatory effect of Dex or TA with TSA Significantly enhanced expression of the marker in all three cell lines. In resting cells, Dex, and TA, in the presence of TSA downregulated caspase 3 expression. These findings support the notion that glucocorticoid actions on apoptosis is mediated, at least in part, through an action on histone acetylation. Finally, histone acetylation was investigated in vivo in two rat models of inflammation and in human subjects with inflammatory bowel disease (IBD). The results showed an increase in histone H4 acetylation lysine specificity of acetylation on K8 and K12 in inflamed tissue and Peyer's patches in animal models and in IBD patients. Whereas H3 acetylation was not elevated to the same extent in tissue and was restricted to the mantle zone of Peyer's patches. In general, the present studies on histone acetylation and inflammation (in animal models and IBD patients) underlined the possibility of a general mechanism linking activation of the transcription factor NFKB with histone acetylation. The ultimate objective of this work is to aid in the understanding of the mechanisms of how deregulation of chromosome structure leads to progression of the disease state. This knowledge may aid in the development of new therapeutic approaches or improved glucocorticoids

Differential patterns of histone acetylation in inflammatory bowel diseases

Post-translational modifications of histones, particularly acetylation, are associated with the regulation of inflammatory gene expression. We used two animal models of inflammation of the bowel and biopsy samples from patients with Crohn's disease (CD) to study the expression of acetylated histones (H) 3 and 4 in inflamed mucosa. Acetylation of histone H4 was significantly elevated in the inflamed mucosa in the trinitrobenzene sulfonic acid model of colitis particularly on lysine residues (K) 8 and 12 in contrast to non-inflamed tissue. In addition, acetylated H4 was localised to inflamed tissue and to Peyer's patches (PP) in dextran sulfate sodium (DSS)-treated rat models. Within the PP, H3 acetylation was detected in the mantle zone whereas H4 acetylation was seen in both the periphery and the germinal centre. Finally, acetylation of H4 was significantly upregulated in inflamed biopsies and PP from patients with CD. Enhanced acetylation of H4K5 and K16 was seen in the PP. These results demonstrate that histone acetylation is associated with inflammation and may provide a novel therapeutic target for mucosal inflammation

Μοντελοποίηση της παροχής αιχμής με τη χρήση UAV- Η περίπτωση του Μαριορέματος στην πλημμύρα του 2016

Στόχος της παρούσας διπλωματικής εργασίας ήταν η μοντελοποίηση της παροχής αιχμής με τη χρήση UAV στο χείμαρρο Μαριόρεμα με αφορμή (και με μελέτη περίπτωσης) το πλημμυρικό φαινόμενο του Σεπτεμβρίου 2016. Αρχικά, έγινε ανάλυση των φαινομένων κατά τη διάρκεια του συγκεκριμένου συμβάντος και αναφορά στην έκταση και τις επιπτώσεις του στην ευρύτερη πληγείσα περιοχή. Για τις ανάγκες της μοντελοποίησης μελετήθηκε η γεωμετρία του αναγλύφου, η τραχύτητα από σημείο σε σημείο της περιοχής μελέτης και η σύγκριση αυτών πριν και μετά την έξαρση του φαινομένου. Τα συγκεκριμένα δεδομένα χαρτογραφήθηκαν με μεγάλη λεπτομέρεια με τη βοήθεια δεδομένων που προέκυψαν από αεροφωτογράφιση με ΣμηΕΑ (drone, UAS). Με τη χρήση του λογισμικού Pix4D mapper έγινε η επεξεργασία των δεδομένων πτήσης που σχεδιάστηκε για αποτύπωση αναγλύφου σε τρεις διαστάσεις (3D model). Από την επεξεργασία αυτή προέκυψαν ορθοφωτοχάρτες υψηλής ανάλυσης, που μας βοήθησαν σε συνδυασμό με τα δεδομένα των παρατηρήσεων υπαίθρου να χαρτογραφηθούν με μεγάλη ακρίβεια η φύση της κοίτης (μητρικό πέτρωμα, αποθέσεις, βλάστηση κ.λπ.) ώστε να υπολογιστεί η τραχύτητα της κοίτης κατά Manning. Χρησιμοποιήσαμε ακόμα το ψηφιακό μοντέλο επιφανείας από τα δεδομένα του UAS για την κατασκευή τομών της κοίτης. Τα δεδομένα της κλίσης της κοίτης και της τομογραφικής τομής χρησιμοποιήθηκαν για τον υπολογισμό της υγρής περιμέτρου, της επιφάνειας διαβροχής και της υδραυλικής ακτίνας, με τα οποία έγινε η εκτίμηση της παροχής αιχμής.The aim of this diploma thesis was to model the inflow peak using UAV in the Mariorema’s flood of September 2016. Initially, an analysis was made of the duration of this event and reference to its extent and its impact on the wider study area. For the modeling needs, the geometry of the embossment, the roughness of the study area before and after. This data mapping was performed by a drone with precision through aerial photography data (UAS). Using Pix4D mapper program lead to the processing of flight data designed for three-dimensional embosses printing (3Dmodel). This handling resulted in high resolution orthophotomaps, which helped to accurately map the stream (rock, deposits, vegetation, etc) to calculate the roughness of the Manning. We also used the digital surface model from the UAS date for the construction of the bed sections. The data of the slope gradient and the tomographic incision were used to calculate the wetter perimeter, wetter surface and the hydraulic radius which assessed the inflow peak

Theophylline Restores Histone Deacetylase Activity and Steroid Responses in COPD Macrophages

Chronic obstructive pulmonary disease (COPD) is a common chronic inflammatory disease of the lungs with little or no response to glucocorticoids and a high level of oxidative stress. Histone deacetylase (HDAC) activity is reduced in cells of cigarette smokers, and low concentrations of theophylline can increase HDAC activity. We measured the effect of theophylline on HDAC activity and inflammatory gene expression in alveolar macrophages (AM) from patients with COPD. AM from normal smokers showed a decrease in HDAC activity compared with normal control subjects, and this was further reduced in COPD patients (51% decrease, P < 0.01). COPD AMs also showed increased basal release of IL-8 and TNF-α, which was poorly suppressed by dexamethasone. Theophylline induced a sixfold increase in HDAC activity in COPD AM lysates and significantly enhanced dexamethasone suppression of induced IL-8 release, an effect that was blocked by the HDAC inhibitor trichostatin A. Therefore, theophylline might restore steroid responsiveness in COPD patients

Differential patterns of histone acetylation in inflammatory bowel diseases

Post-translational modifications of histones, particularly acetylation, are associated with the regulation of inflammatory gene expression. We used two animal models of inflammation of the bowel and biopsy samples from patients with Crohn's disease (CD) to study the expression of acetylated histones (H) 3 and 4 in inflamed mucosa. Acetylation of histone H4 was significantly elevated in the inflamed mucosa in the trinitrobenzene sulfonic acid model of colitis particularly on lysine residues (K) 8 and 12 in contrast to non-inflamed tissue. In addition, acetylated H4 was localised to inflamed tissue and to Peyer's patches (PP) in dextran sulfate sodium (DSS)-treated rat models. Within the PP, H3 acetylation was detected in the mantle zone whereas H4 acetylation was seen in both the periphery and the germinal centre. Finally, acetylation of H4 was significantly upregulated in inflamed biopsies and PP from patients with CD. Enhanced acetylation of H4K5 and K16 was seen in the PP. These results demonstrate that histone acetylation is associated with inflammation and may provide a novel therapeutic target for mucosal inflammation

Genetic testing in professional football: perspectives of key stakeholders

Purpose: Genetic research in football is currently in it’s infancy but is growing rapidly. However, the practical application of genetic testing in football and the views concerning its use are unknown. Thus, the purpose of this study was to assess the current practical application of genetic testing in professional football and provide an insight into the perspectives of key stakeholders (i.e., coaches, practitioners, players). Methods: In total, 122 participants completed an online anonymous survey. This consisted of 21 multiple choice and Likert scale questions, with the option of providing an explanation for each response. Results: Findings revealed genetic testing is rarely utilised by key stakeholders (10%) or their respective organisations (14%). However, three quarters (75%) had the opinion that genetic testing will have great utility in the future. The majority (72%) believed genetic testing should be used for athlete development and injury risk, whilst 35% believed that genetic testing should be utilised for talent identification purposes. However, most key stakeholders viewed their own (89%) and their colleagues’ (79%) knowledge related to genetic testing as insufficient; mainly due to ineffective current communication methods (91%). Most believed educational workshops are required (71%), whilst nearly all (91%) were interested in developing their expertise on the utility of genetic testing. Conclusion: Genetic testing is rarely used within professional football, although key stakeholders anticipate that it will be utilised more in the future. As such, educational support may prove valuable in improving key stakeholder knowledge and the practical application of genetic testing in professional football

Manipulation of dipeptidylpeptidase 10 in mouse and human in vivo and in vitro models indicates a protective role in asthma

We previously identified dipeptidylpeptidase 10 (DPP10) on chromosome 2 as a human asthma susceptibility gene, through positional cloning. Initial association results were confirmed in many subsequent association studies but the functional role of DPP10 in asthma remains unclear. Using the MRC Harwell N-ethyl-N-nitrosourea (ENU) DNA archive, we identified a point mutation in Dpp10 that caused an amino acid change from valine to aspartic acid in the β-propeller region of the protein. Mice carrying this point mutation were recovered and a congenic line was established (Dpp10145D). Macroscopic examination and lung histology revealed no significant differences between wild-type and Dpp10145D/145D mice. However, after house dust mite (HDM) treatment, Dpp10 mutant mice showed significantly increased airway resistance in response to 100 mg/ml methacholine. Total serum IgE levels and bronchoalveolar lavage (BAL) eosinophil counts were significantly higher in homozygotes than in control mice after HDM treatment. DPP10 protein is present in airway epithelial cells and altered expression is observed in both tissue from asthmatic patients and in mice following HDM challenge. Moreover, knockdown of DPP10 in human airway epithelial cells results in altered cytokine responses. These results show that a Dpp10 point mutation leads to increased airway responsiveness following allergen challenge and provide biological evidence to support previous findings from human genetic studies. This article has an associated First Person interview with the first author of the paper

Genetic variations between youth and professional development phase english academy football players

The purpose of this study was to examine differences in the genotype frequency distribution of thirty-three single nucleotide variants (SNVs) between youth development phase (YDP) and professional development phase (PDP) academy football players. One hundred and sixty-six male football players from two Category 1 and Category 3 English academies were examined within their specific age phase: YDP (n = 92; aged 13.84 ± 1.63 years) and PDP (n = 74; aged 18.09 ± 1.51 years). Fisher’s exact tests were used to compare individual genotype frequencies, whereas unweighted and weighted total genotype scores (TGS; TWGS) were computed to assess differences in polygenic profiles. In isolation, the IL6 (rs1800795) G allele was overrepresented in PDP players (90.5%) compared to YDP players (77.2%; p = 0.023), whereby PDP players had nearly three times the odds of possessing a G allele (OR = 2.83, 95% CI: 1.13–7.09). The TGS (p = 0.001) and TWGS (p < 0.001) were significant, but poor, in distinguishing YDP and PDP players (AUC = 0.643–0.694), with PDP players exhibiting an overall more power-orientated polygenic profile. If validated in larger independent youth football cohorts, these findings may have important implications for future studies examining genetic associations in youth football

Core regulatory circuitries in defining cancer cell identity across the malignant spectrum

Gene expression programmes driving cell identity are established by tightly regulated transcription factors that auto- and cross-regulate in a feed-forward manner, forming core regulatory circuitries (CRCs). CRC transcription factors create and engage super-enhancers by recruiting acetylation writers depositing permissive H3K27ac chromatin marks. These super- enhancers are largely associated with BET proteins, including BRD4 that influence higher- order chromatin structure. The orchestration of these events trigger accessibility of RNA polymerase machinery and the imposition of lineage-specific gene expression. In cancers, CRCs drive cell identity by superimposing developmental programmes on a background of genetic alterations. Further, the establishment and maintenance of oncogenic states are reliant on CRCs that drive factors involved in tumour development. Hence, the molecular dissection of CRC components driving cell identity and cancer state can contribute to elucidating mechanisms of diversion from pre-determined developmental programmes and highlight cancer dependencies. These insights can provide valuable opportunities for identifying and repurposing drug targets. In this article, we review the current understanding of CRCs across solid and liquid malignancies and avenues of investigation for drug development efforts. We also review techniques used to understand CRCs and elaborate the indication of discussed CRC transcription factors in the wider context of cancer CRC models

Immunopathogenesis of rheumatoid arthritis

Rheumatoid arthritis (RA) is the most common inflammatory arthropathy. The majority of evidence, derived from genetics, tissue analyses, models, and clinical studies, points to an immune-mediated etiology associated with stromal tissue dysregulation that together propogate chronic inflammation and articular destruction. A pre-RA phase lasting months to years may be characterized by the presence of circulating autoantibodies, increasing concentration and range of inflammatory cytokines and chemokines, and altered metabolism. Clinical disease onset comprises synovitis and systemic comorbidities affecting the vasculature, metabolism, and bone. Targeted immune therapeutics and aggressive treatment strategies have substantially improved clinical outcomes and informed pathogenetic understanding, but no cure as yet exists. Herein we review recent data that support intriguing models of disease pathogenesis. They allude to the possibility of restoration of immunologic homeostasis and thus a state of tolerance associated with drug-free remission. This target represents a bold vision for the future of RA therapeutics