Item Controlled AccessFunctional Imaging the Neonatal Brain with Wearable Diffuse Optical TomographyUchitel, JulieStudying brain development poses significant methodological challenges when working with vulnerable newborn infant populations. Non-invasive and wearable optical brain imaging technologies may allow for the cot-side study of newborn infants in clinical settings. This has particularly important implications for preterm infants in intensive care, for whom traditional neuroimaging techniques are not readily feasible. Wearable high-density diffuse optical tomography (HD-DOT) has emerged as a promising technology in this pursuit. In this thesis, I describe the development and application of wearable HD-DOT for newborn infants in clinical settings. Specifically, this technology is applied to study the features of functional brain connectivity (FC) associated with neonatal sleep states, active sleep and quiet sleep, in both term-born and preterm infants. HD-DOT is also electromechanically combined with electroencephalography (EEG) to concurrently study haemodynamic cortical and electrical cortical brain activity in the preterm infant. As a result of the constraints posed by lock-down conditions and a pause on clinical studies during the COVID-19 pandemic, the HD- DOT device is also used to study resting state functional connectivity (RSFC) in the adult brain in the home setting. Findings from this work demonstrate the potential of wearable HD-DOT, and combined EEG-HD-DOT, for the cot-side study of cerebral function during sleep in newborn infants, as well as at-home neuroimaging of resting-state networks in adults. This work was supervised by: Dr. Topun Austin, Department of Paediatrics, University of Cambridge, and Dr. Robert J. Cooper, Department of Medical Physics and Biomedical Engineering, UCL. Item RestrictedEpigenetic Regulation of Intestinal Epithelial MHC Class I in Health and Inflammatory Bowel DiseaseDennison, Thomas[Restricted] Item EmbargoQuantitative spatial transcriptomics of the developing brainBartels, Theresa; Bartels, Theresa [0000-0003-4100-0608]Developmental and cellular heterogeneity can be studied at the transcriptomic level using RNA sequencing methods, but techniques that quantitatively investigate the spatial dynamics of cell heterogeneity across large tissue areas at a single-cell level are lacking. Applying this capability to the developing brain would enable reconstruction of a full spatial transcriptomic map of neural heterogeneity and the discovery of novel regional neural subtypes with potential functional implications. Such capability would ultimately culminate in the creation of single cell-level tissue atlases with preserved cellular topology. My goal has been to combine single-molecule fluorescence in situ hybridisation (smFISH), automated confocal imaging and analytical tools to derive ‘Quantitative Spatial Transcriptomics (QST)’ for the spatiotemporal characterisation of gene expression across the developing mammalian forebrain. My thesis research has focused in two main areas: 1) Novel methodology for insights into mammalian cortex architecture: I have co-developed (with Dr Omer Bayraktar) an automated, multiplexed smFISH and imaging pipeline for screening brain-wide gene expression at cellular resolution, termed Large-area Spatial Transcriptomics (LaST). LaST allows “mapping back” of transcriptome data from single-cell and single-nuclei RNA sequencing for high-quality in situ validation in a regional qualitative and quantitative manner. In addition, this tool can uncover novel cell heterogeneity and discover unique cell identities based on precisely validated combinations of cellular gene expression. For example, the organisation of neurons into six distinct layers is a hallmark of the mammalian neocortex but it is not known if glial cells also possess any diversified laminar features. Using LaST, the single-neuron expression of layer markers was mapped across the mouse cerebral cortex and identified diversified glutamatergic neuron subclasses and their laminar distribution in the neocortex, including rare transcriptomic types. Moreover, applying LaST to cortical astrocytes, I identified molecular distinctions that were associated with three dorso-ventral astrocyte layers in the somatosensory cortex, which deviate from known layer organisation of glutamatergic neurons. These published results (Bayraktar, Bartels et al., 2020) identify a previously unrecognised spatial complexity to cortical architecture when considering combined patterns of neuron and astrocyte heterogeneity, and are likely to have functional implications. 2) High-resolution transcriptomic developmental biology of Cdkl5: I further developed LaST into QST for cell type-specific quantifications of a single subject gene and analysed the expression of Cyclin-dependent kinase-like 5 (Cdkl5) during forebrain development. CDKL5 mutations cause a severe human neurodevelopmental disorder called CDKL5 deficiency disorder (CDD) that is currently incurable. The expression of CDKL5 in developing neural cells, especially glia, is unclear, resulting in an incomplete understanding of the pathogenesis of the disease. Using QST, I quantified the spatiotemporal expression pattern of Cdkl5 mRNA in developing mouse brain and uncovered novel dynamic patterns of Cdkl5 enrichment. I discovered that Cdkl5 is initially enriched in upper layer neurons perinatally, followed by deep layer neurons during late postnatal development. Using QST and data derived from single-cell RNA sequencing, I found evidence that Cdkl5/CDKL5 is expressed by macroglia during early postnatal development and is enriched in distinct oligodendrocyte transcriptomic types in the mature mouse and human neocortex. An investigation of CDKL5 protein expression in mouse brain development and in human post-mortem tissue is yet to be fully characterised. These data, combined with future investigation of the function of CDKL5 in these cell populations, will improve our understanding of CDD pathogenesis and may uncover novel cellular targets for therapeutic intervention in CDD. In addition, the QST screen provides a quantitative reference map of physiological Cdkl5 expression levels during postnatal development that is instructive for future development of gene and protein replacement therapies. Combined, the novel spatial transcriptomics techniques developed in this thesis identified higher-order cerebral cortex organisation, and allowed characterisation of Cdkl5 gene expression, significantly advancing our understanding of mammalian neocortical development and Cdkl5 gene function therein. Item Open AccessMetabolic phenotypes of infants with normal birth weight, small-for-gestational-age, or after maternal gestational diabetes mellitusOlga, Laurentya; Olga, Laurentya [0000-0002-3562-0598]Numerous studies have associated both under- and overnutrition during early life with long-term metabolic outcomes. Those conditions are typically represented by two groups of infants in animal and human studies: infants born small-for-gestational-age (SGA; reflecting intrauterine undernutrition) and offspring of mothers with gestational diabetes mellitus (OGDM; reflecting intrauterine overnutrition and hyperglycaemia). However, the underlying mechanism behind this phenomenon is still unknown: how these distinct groups can end up with similar metabolic risks, despite having opposite in utero nutritional conditions. This thesis aims to characterise biological similarities and differences across SGA, OGDM, and a control population from the Cambridge Baby Growth Study (CBGS). The CBGS, set up in 2001, is an ongoing longitudinal cohort aiming to examine the ante- and postnatal determinants of infant growth and body composition, including genetic and environmental factors. While SGA infants in CBGS showed typical rapid postnatal growth patterns, the contemporary OGDM cohort showed a distinct trend to that in earlier cohorts, with normal birth weights but reduced adiposity, which was sustained from birth to 24 months. Preliminary analyses of infant capillary blood spot profiles suggested that pre- and postnatal exposures reflected in SGA and OGDM may share common hormonal and lipidomic signatures during early infancy, independent of feeding practice and other confounding factors. In a CBGS breastmilk (BM) study, higher BM intake volume at 6 weeks conferred protection against subsequent rapid weight gain. Analyses of BM macronutrients also suggested that carbohydrate and protein intakes may have functional relevance to later infant growth and adiposity. This work has characterised in detail the effects of antenatal and postnatal nutritional factors on infant growth, body composition and biochemical profiles. The early infancy metabolic signatures identified here may reflect the continuum of early programming from pre- to early postnatal and might be potentially linked to future metabolic risks. Item Open AccessThe relationship between enteral nutrition, energy metabolism and gut homeostasis during the course of critical illness.(2019-06-04) Zaher, SaraNutrition has an indirect effect on the gastrointestinal function of the host and thereby on health, mainly by influencing the composition and activity of the human gut microbiota. The aim of this PhD project was to investigate the effect of nutrition as a factor affecting the intestinal microbiome-host relationship in critically ill children and whether it has an impact on clinical disease severity. The balance between requirement and delivery of energy and macronutrients was assessed in a cohort of 124 critically ill children. Then an integrated approach of metataxonomics and metabolomics analysis was undertaken to examine how feeding during critical illness affects the gut-host relationship. Collection of faecal samples was required for the assessment of faecal calprotectin, gut microbiota and their metabolites while serum samples were used for the analysis of inflammatory cytokines, and intestinal injury biomarkers. Overall this project has recorded a cross-link between feed, gut homeostasis with systemic inflammation and host metabolism. Within the feed delivered, fat delivery was often above requirements compared to protein and carbohydrate. In addition, both protein deficit and higher delivery of fat were associated with elevation in the levels of pro-inflammatory cytokines. The results also showed that abnormalities in gut health biomarkers were associated with elevation in inflammatory cytokines. Finally this study also recorded a profound loss of diversity in the faecal microbiome of critically ill children. This was associated with the loss of key commensal species and increased levels of opportunistic pathogens. Consequently resulted in reduced functionality of the gut microbiome manifested by reduced production of SCFAs and abnormalities in BAs metabolism. The current study showed for the first time that energy underfeeding appeared to influence the microbial composition of critically ill children. In conclusion this work provided an insight about the potential contribution of nutrition as a factor to improve the disease state in critically ill children, if targeted to modulate gut microbiome and host response to critical illness. Item Open AccessThe Artificial Pancreas in Children and Adolescents with Type 1 Diabetes: Bringing Closed-Loop Home(2019-04-27) Tauschmann, Martin; Tauschmann, Martin [0000-0002-2305-2490]Type 1 diabetes is one of the most common chronic conditions in childhood and adolescence. Despite ongoing development of more physiological insulin preparations, recent advancements in insulin pump technology and more accurate blood glucose monitoring, in clinical practice it remains challenging to achieve normoglycaemia whilst reducing the risk of hypoglycaemia, particularly in young people with type 1 diabetes. Closed-loop insulin delivery (the artificial pancreas) is an emerging technology gradually progressing from bench to clinical practice. Closed-loop systems combine glucose sensing with computer-based algorithm informed insulin delivery to provide real-time glucose-responsive insulin administration. The key objective of my thesis is to evaluate the safety, efficacy and utility of closed-loop insulin delivery in children and adolescents with type 1 diabetes outside of the research facility setting. Results of five clinical trials are presented in the main chapters of this thesis. In a mechanistic study, the impact of glucose sensor operation duration on efficacy of overnight closed-loop was investigated comparing closed-loop performance on day 1 of sensor insertion to day 3 to 4 of sensor. Twelve adolescents with type 1 diabetes attended the research facility for two overnight visits. The sequence of the interventions was random. Despite differences in sensor accuracy, overnight CL glucose control informed by sensor glucose on day 1 or day 3-4 after sensor insertion was comparable. The model predictive controller appears to mitigate against sensor inaccuracies. In home settings, overnight closed-loop application was evaluated over three months in 25 children and adolescents with type 1 diabetes aged six to 18 years. The study was conducted at three centres in the UK and adopted a randomised cross-over design. Compared to sensor-augmented pump therapy, overnight home use of closed-loop increased the proportion of time sensor glucose was in target, and reduced mean glucose and hypoglycaemia. Two randomised crossover studies evaluated the safety and efficacy of day-and-night hybrid closed-loop insulin delivery in young people with type 1 diabetes aged 10 to 18 years over seven days, and 21 days, respectively. A total of 24 subjects were enrolled in this single centre trial. Free-living home use of day-and-night closed-loop in suboptimally controlled adolescents with type 1 diabetes was safe, and improved glucose control without increasing the risk of hypoglycaemia. Finally, closed-loop technology was assessed in five very young children (aged one to seven years) with type 1 diabetes in a two-period, crossover study. Closed-loop was used during both 3-week intervention periods, either with standard strength insulin (U100), or with diluted insulin (U20). The order of intervention was random. Free-living home use of day-and-night hybrid closed-loop in very young children with type 1 diabetes was feasible and safe. Glucose control was comparable during both intervention periods. Thus, use of diluted insulin during closed-loop insulin delivery might not be of additional benefit in this population. In conclusion, studies conducted as part of my thesis demonstrate that use of hybrid closed-loop insulin delivery systems in children and adolescents aged one to 18 years in free daily living without remote monitoring or supervision is feasible, safe and effective. My work supports the progression of this technology from research to mainstream clinical practice.