Sortilin: A Protein Involved in Ldl Metabolism and Atherosclerosis

Genome-wide association studies (GWAS) have been used to identify novel genes and loci that contribute to lipid traits and coronary heart disease (CHD) in a causal manner. A locus on chromosome 1p13, which harbors the gene sortilin-1 (SORT1) encoding the protein sortilin is the locus in the human genome with the strongest association with low-density lipoprotein cholesterol (LDL-C) and is also one of the strongest loci associated with CHD. Homozygosity for the minor allele haplotype at 1p13 is associated with a \u3e10 fold increase in hepatic SORT1 expression, a mean 16 mg/dL reduction in plasma LDL-C, and a 40% reduction in CHD risk. Sortilin has been extensively studied in the central nervous system, where it traffics multiple ligands from the Golgi apparatus to the lysosome and also serves as a cell surface endocytosis receptor for a variety of ligands. However, the role of sortilin in other cell types, most notably hepatocytes and macrophages, which are key regulators of lipid metabolism and atherosclerosis development, has not been well studied. Through a series of overexpression and mutagenesis studies in cells and mice, the Rader lab has previously shown that increased sortilin expression in liver reduces plasma LDL-C both by promoting the presecretory lysosomal degradation of the LDL precursor very-low density lipoprotein (VLDL) and by serving as an endocytosis receptor for LDL. The Rader lab has also shown that total body Sort1 deficiency is associated with compromised LDL clearance consistent with overexpression studies; however, it is also associated with a paradoxical reduction in VLDL secretion. Using a variety of liver specific Sort1 deficiency models, as well as reconstitution and mutagenesis studies, I showed that liver specificity is not responsible for the secretion paradox and instead demonstrated that sortilin plays a dual role in VLDL trafficking, serving as a chaperone that facilitates VLDL secretion as well as a transporter that promotes the presecretory degradation of VLDL, depending on the conditions and level of sortilin expression. Sortilin is strongly associated both with LDL-C levels and with CHD/atherosclerosis. Atherosclerotic cardiovascular disease is driven by elevated LDL-C, thus it is tempting to speculate that the strong association of the SORT1 locus with atherosclerosis is due solely to the LDL-C association. Because sortilin is expressed in macrophages, I hypothesized that macrophage sortilin might influence atherogenesis. Through careful interrogation of the role of sortilin in macrophages, I demonstrated that sortilin plays a role in the development of atherosclerosis independent of plasma LDL-C levels. Specifically, I showed that sortilin serves as an endocytosis receptor for LDL on macrophages, and this represents a physiologically important pathway by which LDL cholesterol enters macrophages and contributes to foam cell formation and atherosclerosis. This work increases our understanding of the role of hepatic and macrophage sortilin in LDL metabolism and atherogenesis, and provides insight into the relationship of the SORT1 locus with LDL-C levels and CHD risk

A structural method for the design of fault tolerant distributed control systems

Distributed digital control systems provide alternatives to conventional, centralised digital control systems. Typically, a modern distributed control system will comprise a multi-processor or network of processors, a communications network, an associated set of sensors and actuators, and the systems and applications software. This thesis addresses the problem of how to design robust decentralised control systems, such as those used to control event-driven, real-time processes in time-critical environments. Emphasis is placed on studying the dynamical behaviour of a system and identifying ways of partitioning the system so that it may be controlled in a distributed manner. A structural partitioning technique is adopted which makes use of natural physical sub-processes in the system, which are then mapped into the software processes to control the system. However, communications are required between the processes because of the disjoint nature of the distributed (i.e. partitioned) state of the physical system. The structural partitioning technique, and recent developments in the theory of potential controllability and observability of a system, are the basis for the design of controllers. In particular, the method is used to derive a decentralised estimate of the state vector for a continuous-time system. The work is also extended to derive a distributed estimate for a discrete-time system. Emphasis is also given to the role of communications in the distributed control of processes and to the partitioning technique necessary to design distributed and decentralised systems with resilient structures. A method is presented for the systematic identification of necessary communications for distributed control. It is also shwon that the structural partitions can be used directly in the design of software fault tolerant concurrent controllers. In particular, the structural partition can be used to identify the boundary of the conversation which can be used to protect a specific part of the system. In addition, for certain classes of system, the partitions can be used to identify processes which may be dynamically reconfigured in the event of a fault. These methods should be of use in the design of robust distributed systems

Experimental study of transient properties of a wide pin diode

The purpose of this thesis has been to study some of the properties of the hole-electron plasma in the i-layer of a wide p-i-n diode, particularly phenomena associated with the build-up of plasma in a diode, initially at equilibrium, excited with various voltage signals. The investigations of changes in plasma density with time have made use of the fact that this quantity is almost linearly related to the conductance of the diode. Thus the study was carried out by observing the transmitted current resulting from the application of various signals

An Approximate Analytical Solution of the Burger's Equation for Longitudinal Dispersion Phenomenon Arising in Fluid Flow through Porous Medium

The present paper discusses the longitudinal dispersion phenomenon in miscible fluid flow through porous medium. The mathematical formulation yields a nonlinear partial differential equation in the form of Burger's equation. An approximate analytical solution of the Burger's equation for longitudinal dispersion phenomenon has been obtained. Homotopy analysis method is adopted to solve this equation with suitable boundary conditions. The numerical interpretation of solution has been obtained at distancex for a given timet. The graphical interpretation of solution has been also given by Mathematica software

Growth of human breast cells in primary culture

Human breast tumours were enzymatically disaggregated with collagenase and established in primary culture on a variety of feeder layers of mitomycin-C treated mouse embryo fibroblasts. The NIH-3T3 and STO cell lines proved the most successful in promoting the growth of primary breast cultures (successful cultures from approximately 80% of breast tumours)

Circulating Tumour DNA in Localised Urological Cancers

There is a need for informative biomarkers in localised urological cancers. At present, no method can accurately distinguish between indolent and aggressive prostate cancers, and men often require repeated biopsies. Patients with muscle invasive bladder cancer undergo neo-adjuvant chemotherapy (NAC) to improve survival. However many do not respond to NAC, delaying definitive treatment. Cell-free mutant DNA (mutDNA) analysis represents an opportunity for non-invasive monitoring of cancer through tumour genome analysis. MutDNA derived from plasma can monitor tumour burden. There is emerging evidence that mutDNA can identify mutations from multiple clones and is abundant in adjacent body fluids. This work explores the utility of plasma and urinary mutDNA in localised prostate and bladder cancers. This thesis describes the optimisation of urinary mutDNA analysis by assessing urinary DNA processing and extraction methods using healthy volunteer and bladder cancer patient urine samples. Primer panels were designed and validated to target frequently mutated regions in prostate and bladder cancers, as well as for analysis of patient-specific mutations. Sequencing-based methods and dPCR were employed to analyse clinical samples including plasma and urine, to detect and quantify mutDNA. Molecular and clinical data were integrated to explore potential areas of application of mutDNA analysis. For bladder cancer, mutDNA was analysed from liquid-biopsy samples including plasma, cell pellets from urine and urine supernatant from multiple time-points of 17 MIBC patients undergoing NAC. I showed that mutDNA was more frequently detected and was present at higher AFs in urine compared to plasma samples. Of potential clinical relevance, I showed that the presence of mutDNA after starting NAC was associated with disease recurrence. This original contribution to knowledge could offer patients an opportunity to expedite surgical resection in a timely manner, if corroborated in large-scale trials. For prostate cancer, a TP53 specific panel was applied to men with metastatic disease, to demonstrate that clones containing TP53 mutations, which are dominant in at the metastatic stage were present in historical prostatectomy samples taken when then patient was believed to have localised disease only. Furthermore, I showed that these TP53 mutations could be detected at the localised stage of disease. To investigate the ability of mutDNA detection private clonal mutations I developed a method for higher sensitivity analysis (MRD-Seq). This was applied to a clinical cohort of 2 men with multi-focal localised prostate cancer to demonstrate the though the overall levels of mutDNA is low, private clonal mutations may be detectable. Taken together, these original contributions to knowledge could allow for less invasive surveillance of men with low risk prostate cancer and warrants further investigation. In this thesis, I used a range of molecular methods were applied to small cohorts of clinical samples from patients with urological malignancies, in an exploratory analysis. The molecular data was analysed in conjunction with clinical information to draw hypotheses on the biology and natural history of these cancer, and to suggest possible utility of mutDNA analysis in their clinical management. Some of the findings suggest areas of potential utility, which merit further validation or investigation in larger cohorts or clinical studies.Cambridge Cancer Centre Addenbrookes Charitable Trust Royal College of Surgeon

Hyperglycemia induces differential change in oxidative stress at gene expression and functional levels in HUVEC and HMVEC

Abstract Background Endothelial dysfunction precedes pathogenesis of vascular complications in diabetes. In recent years, the mechanisms of endothelial dysfunction were investigated to outline strategies for its treatment. However, the therapies for dysfunctional endothelium resulted in multiple clinical trial failures and remain elusive. There is a need for defining hyperglycemia-induced endothelial dysfunction with both generic and specific dysfunctional changes in endothelial cells (EC) using a systems approach. In this study, we investigated hyperglycemia-induced endothelial dysfunction in HUVEC and HMVEC. We investigated hyperglycemia-induced functional changes (superoxide (O2‾), and hydrogen peroxide (H2O2) production and mitochondrial membrane polarization) and gene expression fingerprints of related enzymes (nitric oxide synthase, NAD(P)H oxidase, and reactive oxygen species (ROS) neutralizing enzymes) in both ECs. Method Gene expression of NOS2, NOS3, NOX4, CYBA, UCP1, CAT, TXNRD1, TXNRD2, GPX1, NOX1, SOD1, SOD2, PRDX1, 18s, and RPLP0 were measured using real-time PCR. O2‾ production was measured with dihydroethidium (DHE) fluorescence measurement. H2O2 production was measured using Amplex Red assay. Mitochondrial membrane polarization was measured using JC-10 based fluorescence measurement. Results We showed that the O2‾ levels increased similarly in both ECs with hyperglycemia. However, these endothelial cells showed significantly different underlying gene expression profile, H2O2 production and mitochondrial membrane polarization. In HUVEC, hyperglycemia increased H2O2 production, and hyperpolarized mitochondrial membrane. ROS neutralizing enzymes SOD2 and CAT gene expression were downregulated. In contrast, there was an upregulation of nitric oxide synthase and NAD(P)H oxidase and a depolarization of mitochondrial membrane in HMVEC. In addition, ROS neutralizing enzymes SOD1, GPX1, TXNRD1 and TXNRD2 gene expression were significantly upregulated in high glucose treated HMVEC. Conclusion Our findings highlighted a unique framework for hyperglycemia-induced endothelial dysfunction. We showed that multiple pathways are differentially affected in these endothelial cells in hyperglycemia. High occurrences of gene expression changes in HMVEC in this study supports the hypothesis that microvasculature precedes macrovasculature in epigenetic regulation forming vascular metabolic memory. Identifying genomic phenotype and corresponding functional changes in hyperglycemic endothelial dysfunction will provide a suitable systems biology approach for understanding underlying mechanisms and possible effective therapeutic intervention

Theoretical Approach in Reducing Total Network Power Consumption by Utilizing 5G Technologies

We expect future 5G network to have low power consumption and minimal delay, as well as a more flexible and secure architecture. Most of the research is concerned primarily with increasing energy efficiency and decreasing latency. Implementation of Software Defined Mobile Networking (SDMN) to control the range of cell site communication and device-to-device (D2D) technologies to enable the devices on the network to behave as routers between other devices and the cell site has the potential to significantly reduce the overall power consumption in the mobile network. Our research provides a theoretical approach for such model and shows that its implementation will increase energy efficiency in 5G