Exploiting Weak Shape Constraints to Segment Capillary Images in Microangiopathy

Microangiopathy is one form of pathology associated with peripheral neuropathy in diabetes. Capillaries imaged by electron microscopy show a complex textured appearance, which makes segmentation difficult. Considerable variation occurs among boundaries manually positioned by human experts. Detection of region boundaries using Active Contour Models has proved impractical due to the existence of confusing image evidence in the vicinity of these boundaries. Despite the fact that the shapes have no identifying landmarks, the weak constraints imposed by statistical shape modelling combined with genetic search can provide accurate segmentations

Glaucoma

This book addresses the basic and clinical science of glaucomas, a group of diseases that affect the optic nerve and visual fields and is usually accompanied by increased intraocular pressure. The book incorporates the latest development as well as future perspectives in glaucoma, since it has expedited publication. It is aimed for specialists in glaucoma, researchers, general ophthalmologists and trainees to increase knowledge and encourage further progress in understanding and managing these complicated diseases

Role of complement genetic variants in inflammatory diseases by an interactive database and protein structure modelling

The rare diseases atypical haemolytic uraemic syndrome (aHUS) and C3 glomerulopathy (C3G) are associated with dysregulation of complement activation. It is unclear which genes most frequently predispose to aHUS or C3G. Accordingly, a six- centre analysis of 610 rare genetic variants in 13 mostly complement genes from >3500 patients with aHUS and C3G was performed. A new interactive Database of Complement Gene Variants was developed to extract allele frequencies for these 13 genes using the Exome Aggregation Consortium server as the reference genome. For aHUS, significantly more protein-altering rare variation was found in the five genes CFH, CFI, CD46, C3 and DGKE than in ExAC. For C3G, an association was only found for rare variants in C3 and the N-terminal C3b-binding or C-terminal non-surface-associated regions of factor H (FH). FH is the major regulator of C3b and its Tyr402His polymorphism is an age-related macular degeneration risk-factor. To better understand FH complement binding, the solution structures of both allotypes were studied. Starting from known FH short complement regulator domains and glycan structures, small angle X-ray scattering data were fitted using Monte Carlo methods to determine atomistic structures for monomeric FH. The analysis of 29,715 physically realistic but randomised FH conformations resulted in 100 similar best-fit FH structures for each allotype. Two distinct molecular structures resulted; an extended N-terminal domain arrangement with a folded-back C-terminus, or an extended C-terminus and folded-back N-terminus. To clarify FH functional roles in host protection, crystal structures for the FH complexes with C3b and C3dg revealed that the extended N-terminal conformation accounted for C3b fluid phase regulation, the extended C-terminal conformation accounted for C3d binding, and both conformations accounted for bivalent FH binding to the host cell-surface. Finally, statistical analyses indicated that the structural location of rare variants in complement may predict the occurrences of aHUS or C3G

Smoking and Second Hand Smoking in Adolescents with Chronic Kidney Disease: A Report from the Chronic Kidney Disease in Children (CKiD) Cohort Study

The goal of this study was to determine the prevalence of smoking and second hand smoking [SHS] in adolescents with CKD and their relationship to baseline parameters at enrollment in the CKiD, observational cohort study of 600 children (aged 1-16 yrs) with Schwartz estimated GFR of 30-90 ml/min/1.73m2. 239 adolescents had self-report survey data on smoking and SHS exposure: 21 [9%] subjects had “ever” smoked a cigarette. Among them, 4 were current and 17 were former smokers. Hypertension was more prevalent in those that had “ever” smoked a cigarette (42%) compared to non-smokers (9%), p\u3c0.01. Among 218 non-smokers, 130 (59%) were male, 142 (65%) were Caucasian; 60 (28%) reported SHS exposure compared to 158 (72%) with no exposure. Non-smoker adolescents with SHS exposure were compared to those without SHS exposure. There was no racial, age, or gender differences between both groups. Baseline creatinine, diastolic hypertension, C reactive protein, lipid profile, GFR and hemoglobin were not statistically different. Significantly higher protein to creatinine ratio (0.90 vs. 0.53, p\u3c0.01) was observed in those exposed to SHS compared to those not exposed. Exposed adolescents were heavier than non-exposed adolescents (85th percentile vs. 55th percentile for BMI, p\u3c 0.01). Uncontrolled casual systolic hypertension was twice as prevalent among those exposed to SHS (16%) compared to those not exposed to SHS (7%), though the difference was not statistically significant (p= 0.07). Adjusted multivariate regression analysis [OR (95% CI)] showed that increased protein to creatinine ratio [1.34 (1.03, 1.75)] and higher BMI [1.14 (1.02, 1.29)] were independently associated with exposure to SHS among non-smoker adolescents. These results reveal that among adolescents with CKD, cigarette use is low and SHS is highly prevalent. The association of smoking with hypertension and SHS with increased proteinuria suggests a possible role of these factors in CKD progression and cardiovascular outcomes

The development of microfluidic based processes

Doctor of Science (DSc) thesis.Full version unavailable due to 3rd party copyright restrictions

The spatial dynamics of insulin-regulated GLUT4 dispersal

Insulin regulates glucose homeostasis by stimulation of glucose transport into adipose and muscle tissues through the regulated trafficking of glucose transporter 4 (GLUT4). In response to insulin GLUT4 rapidly translocates from intracellular storage sites to the plasma membrane where it facilitates glucose uptake. Significant impairments in glucose transport and GLUT4 trafficking are a major hallmark of diabetes mellitus type II. Recent advances in light microscopy techniques enabled the study of GLUT4 dynamics in the plasma membrane and it was reported that the transporter was clustered in the basal state and insulin stimulation resulted in GLUT4 dispersal. The main aim of this study was to develop a microscopy-based assay to study and quantify insulin-stimulated GLUT4 dispersal dynamics in the plasma membrane. Insulin-stimulated GLUT4 dispersal has only been observed in adipocytes and therefore we have chosen this model as a starting point to investigate the molecular mechanisms behind GLUT4 clustering and dispersal. We explored a range of cluster analysis methods to find the most suitable way to quantify GLUT4 clustering dynamics. Furthermore, this project aimed to optimise super resolution imaging in a variety of cell culture models to determine whether insulin-stimulated GLUT4 dispersal operates in skeletal and cardiac muscle and whether this process is affected by disease. Using a range of approaches we showed that insulin results in GLUT4 translocation and dispersal within the plasma membrane of 3T3-L1 adipocytes. We found that AMPK activation attenuated insulin-stimulated glucose uptake in 3T3-L1 adipocytes and also GLUT4 dispersal. It was observed that cholesterol depletion resulted in increased glucose uptake rates and GLUT4 clustering. Knock down of the membrane-localised protein EFR3 that has previously been shown to be involved in glucose uptake resulted in disruption of GLUT4 dispersal in adipocytes. We also found that HeLa cells show similar insulin-stimulated GLUT4 dispersal as adipocytes and suggest that HeLa cells are a suitable experimental model for initial studies of GLUT4 trafficking and dispersal. Chronic insulin treatment was observed to induce a state of cellular insulin resistance in 3T3-L1 adipocytes and resulted in reduced GLUT4 translocation and a more clustered GLUT4 configuration for both basal and insulin-stimulated cells. This indicates that insulin resistance affects intracellular GLUT4 trafficking pathways as well as the organization of the transporter within the plasma membrane in adipocytes. Moreover, we found a negative correlation between adipocyte cell area and insulin-stimulated GLUT4 translocation. We also report that insulin did not stimulate the reorganisation of the transferrin receptor in the plasma membrane of HeLa cells suggesting that insulin-stimulated GLUT4 dispersal did not originate from endosomal compartments in HeLa cells and that this observed effect may be specific for GLUT4. Finally, we observed that insulin did not affect GLUT4 distribution in the membrane of a commercially available model of skeletal muscle from healthy and diabetic donors. Sortilin is a sorting receptor involved in the formation of GLUT4 containing vesicles and levels of this protein were found to be reduced in skeletal muscle myotubes derived from a diabetic donor. Finally, we discovered that insulin stimulated GLUT4 dispersal also operates in stem cell-derived cardiomyocytes and have investigated GLUT4 dispersal in a variety of in vitro models of cardiac muscle tissue. Taken together, this thesis has detailed several novel findings regarding the regulation of GLUT4 clustering in adipose and muscle tissues. A robust assay to measure GLUT4 dispersal has been established and molecular mechanisms behind the observed GLUT4 clustering dynamics have been described in adipocytes. Furthermore GLUT4 clustering was characterised in several cell culture models of skeletal and cardiac muscle for the first time