7,356 research outputs found

    Identifying alterations in adipose tissue-derived islet GPCR peptide ligand mRNAs in obesity: implications for islet function

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    In addition to acting as an energy reservoir, white adipose tissue is a vital endocrine organ involved in the modulation of cellular function and the maintenance of metabolic homeostasis through the synthesis and secretion of peptides, known as adipokines. It is known that some of these secretory peptides play important regulatory roles in glycaemic control by acting directly on islet β-cells or on insulin-sensitive tissues. Excess adiposity causes alterations in the circulating levels of some adipokines which, depending on their mode of action, can have pro-inflammatory, pro-diabetic or anti-inflammatory, anti-diabetic properties. Some adipokines that are known to act at β-cells have actions that are transduced by binding to G protein- coupled receptors (GPCRs). This large family of receptors represents ~35% of all current drug targets for the treatment of a wide range of diseases, including type 2 diabetes (T2D). Islets express ~300 GPCRs, yet only one islet GPCR is currently directly targeted for T2D treatment. This deficit represents a therapeutic gap that could be filled by the identification of adipose tissue-derived islet GPCR peptide ligands that increase insulin secretion and overall β-cell function. Thus, by defining their mechanisms of action, there is potential for the development of new pharmacotherapies for T2D. Therefore, this thesis describes experiments which aimed to compare the expression profiles of adipose tissue-derived islet GPCR peptide ligand mRNAs under lean and obese conditions, and to characterise the functional effects of a selected candidate of interest on islet cells. Visceral fat depots were retrieved from high-fat diet-induced and genetically obese mouse models, and from human participants. Fat pads were either processed as whole tissue, or mature adipocyte cells were separated from the stromal vascular fraction (SVF) which contains several other cell populations, including preadipocytes and macrophages. The expression levels of 155 islet GPCR peptide ligand mRNAs in whole adipose tissue or in isolated mature adipocytes were quantified using optimised RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocols. Comparisons between lean and obese states in mice models and humans revealed significant modifications in the expression levels of several adipokine mRNAs. As expected, mRNAs encoding the positive control genes, Lep and AdipoQ were quantifiable, with the expression of Lep mRNA increasing and that of AdipoQ mRNA decreasing in obesity. Expression of Ccl4 mRNA, encoding chemokine (C-C motif) ligand 4, was significantly upregulated in whole adipose tissue across all models of obesity compared to their lean counterparts. This coincided with elevated circulating Ccl4 peptide levels. This increase was not replicated in isolated mature adipocytes, indicating that the source of upregulated Ccl4 expression in obesity was the SVF of adipose tissue. Based on this significant increase in Ccl4 mRNA expression within visceral fat and its undetermined effects on β-cell function, Ccl4 was selected for further investigation in MIN6 β-cells and mouse islets. PRESTO-Tango β-arrestin reporter assays were performed to determine which GPCRs were activated by exogenous Ccl4. Experiments using HTLA cells expressing a protease-tagged β- arrestin and transfected with GPCR plasmids of interest indicated that 100ng/mL Ccl4 significantly activated Cxcr1 and Cxcr5, but it was not an agonist at the previously identified Ccl4-target GPCRs Ccr1, Ccr2, Ccr5, Ccr9 and Ackr2. RNA extraction and RT-qPCR experiments using MIN6 β-cells and primary islets from lean mice revealed the expression of Cxcr5 mRNA in mouse islets, but it was absent in MIN6 β-cells. The remaining putative Ccl4 receptors (Ccr1, Ccr2, Ccr5, Ccr9, Cxcr1 and Ackr2) were either absent or present at trace levels in mouse islets and MIN6 β-cells. Recombinant mouse Ccl4 protein was used for functional experiments at concentrations of 5, 10, 50 and 100ng/mL, based on previous reports of biological activities at these concentrations. Trypan blue exclusion testing was initially performed to assess the effect of exogenous Ccl4 on MIN6 β-cell viability and these experiments indicated that all concentrations (5-100ng/mL) were well-tolerated. Since β-cells have a low basal rate of apoptosis, cell death was induced by exposure to the saturated free fatty acid, palmitate, or by a cocktail of pro-inflammatory cytokines (interleukin-1β, tumour necrosis factor-α and interferon-γ). In MIN6 β-cells, Ccl4 demonstrated concentration-dependent protective effects against palmitate-induced and cytokine-induced apoptosis. Conversely, while palmitate and cytokines also increased apoptosis of mouse islets, Ccl4 did not protect islets from either inducer. Quantification of bromodeoxyuridine (BrdU) incorporation into β-cell DNA indicated that Ccl4 caused a concentration-dependent reduction in proliferation of MIN6 β-cells in response to 10% fetal bovine serum (FBS). In contrast, immunohistochemical quantification of Ki67-positive mouse islet β-cells showed no differences in β-cell proliferation between control- and Ccl4-treated islets. Whilst the number of β-cells and δ-cells were unaffected, α- cells were significantly depleted by Ccl4 treatment. Exogenous Ccl4 had no effect on nutrient- stimulated insulin secretion from both MIN6 β-cells and primary mouse islets. The 3T3-L1 preadipocyte cell line was used to assess potential Ccl4-mediated paracrine and/or autocrine signalling within adipose tissue. Ccl4 did not alter the mRNA expression of Pparγ, a master regulator of adipocyte differentiation, but did significantly downregulate the mRNA expression of the crucial adipogenic gene, adiponectin. Oil Red O staining and Western blotting were performed to assess lipid accumulation, and insulin and lipolytic signalling, respectively, and these experiments indicated that the observed Ccl4-induced decrease in adiponectin expression failed to correlate with any changes in adipocyte function. In summary, these data demonstrated anti-apoptotic and anti-proliferative actions of the adipokine, Ccl4, on MIN6 β-cells that were not replicated in mouse islets. The absence of any anti-apoptotic, insulin secretory and/or pro-proliferative effects of Ccl4 in islet β-cells suggests that it is unlikely to play a role in regulating β-cell function via crosstalk between adipose tissue and islets. The divergent functional effects highlight that whilst MIN6 cells are a useful primary β-cell surrogate for some studies, primary islets should always be used to confirm physiological relevance. On the other hand, significant α-cell depletion following Ccl4 treatment suggests a cell-specific function within the islets. Furthermore, Ccl4 impaired adiponectin mRNA expression in adipocytes, although, how adipocyte function is affected as a result requires further investigation. Collectively, these data have contributed increased understanding of the role of obesity in modifying the expression of adipose tissue-derived islet GPCR peptide ligands

    Biomarkers in acute ischemic stroke

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    Beam scanning by liquid-crystal biasing in a modified SIW structure

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    A fixed-frequency beam-scanning 1D antenna based on Liquid Crystals (LCs) is designed for application in 2D scanning with lateral alignment. The 2D array environment imposes full decoupling of adjacent 1D antennas, which often conflicts with the LC requirement of DC biasing: the proposed design accommodates both. The LC medium is placed inside a Substrate Integrated Waveguide (SIW) modified to work as a Groove Gap Waveguide, with radiating slots etched on the upper broad wall, that radiates as a Leaky-Wave Antenna (LWA). This allows effective application of the DC bias voltage needed for tuning the LCs. At the same time, the RF field remains laterally confined, enabling the possibility to lay several antennas in parallel and achieve 2D beam scanning. The design is validated by simulation employing the actual properties of a commercial LC medium

    Advances in performance and automation of a single ytterbium ion optical clock

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    While the SI second is currently defined in terms of a microwave transition frequency in caesium, atomic clocks based on an optical transition are currently outperforming caesium clocks by up to two orders of magnitude. In order to fully exploit the potential accuracy achievable by optical clocks, the SI second needs to be redefined in terms of an optical frequency standard. The ¹⁷¹Yb⁺ ion is an excellent candidate thanks to the extremely narrow linewidth of its electric octupole (E3) transition and its particular insensitivity to external perturbations. This thesis is focused on the ytterbium ion optical clock at the National Physical Laboratory (NPL), consisting of a single ¹⁷¹Yb⁺ ion trapped in a radio frequency (RF) Paul trap and probed by ultrastable 467-nm light to excite the E3 transition. Improved measurement methods were developed for the evaluation of several systematic frequency shifts. In particular, the electric quadrupole shift, which used to be the leading source of uncertainty, can now be directly measured with an accuracy in the low parts in 10¹⁸. A great focus was put on the automation of several aspects of the experiment. Because all optical clocks generally require a lot of maintenance and attention during their operation, many experimental routines were automated in order to minimise the requirement for human intervention. Furthermore, the analysis of almost all systematic shifts was automated, requiring minimal manual input so that shifts could be evaluated on the fly. Finally, a generalised framework was developed for the automatic evaluation of the absolute frequency of the optical clock via the International Atomic Time (TAI). In order to increase the confidence in the level of performance of the ytterbium ion optical clock, international clock comparison campaigns are regularly carried out. Between 2019 and 2022, several results were produced: two absolute frequency measurements via TAI with an uncertainty at the 1 × 10⁻¹⁵ level; two local frequency ratio measurements between ¹⁷¹Yb⁺ (E3) and ⁸⁷Sr with an uncertainty in the low parts in 10¹⁷; three uncertainty budgets at the parts in 10¹⁸ level; and one measurement of the ratio of the octupole and quadrupole optical clock transitions in ¹⁷¹Yb⁺ with an uncertainty of 1.5 × 10⁻¹⁶. All of these results are shown to be consistent with each other and in good agreement with the literature. Furthermore, a prototype optically-steered time scale was successfully demonstrated for the first time at NPL with the contribution of both the ¹⁷¹Yb⁺ and ⁸⁷Sr optical clocks.Open Acces

    Performance and degradation studies of IrOx for polymer electrolyte membrane water electrolyser applications

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    In order to enable the scale-up of the proton exchange membrane (PEM) electrolyser technology to the terawatt level, improvements in anode catalyst utilisation are necessary. State of the art PEM electrolysers typically use IrOx to catalyse the oxygen evolution at the anode; however, further improvements in iridium utilisation need to be made without compromising performance or device lifetime. The research community has only recently started to attempt systematic benchmarking of catalyst stability. Short term electrochemical methods alone are insufficient to predict catalyst degradation; they can both underestimate and overestimate catalyst durability. In this work, detailed methods to track trends in catalyst stability using conventional techniques such as rotating disk electrode studies are conducted and supported by complementary techniques. These complementary techniques include inductively coupled plasma – mass spectrometry to track performance and stability during accelerated stress testing. These methods were then assessed by testing a series of IrOx nanoparticles both commercially obtained and synthesized via a variant of the Adams Fusion method. Catalysts synthesized via Adams Fusion method demonstrated significant increases in stability with approximately an order of magnitude difference in dissolved Ir observed between those synthesized at 400 oC and 500 oC. This correlates with change between amorphous (400 oC) and crystalline (500 oC, 600 oC) structure with less significant improvements in stability being seen between 500 oC and 600 oC. Increasing annealing temperature also correlated with reduced surface area and reduced activity. The comparison of different lower potential limits during stability testing also demonstrated increased dissolution upon decreasing lower potential limit. Therefore, the observations indicate that electrochemical reduction plays a large role in the heightened dissolution observed from potential cycling. Several operando techniques are also demonstrated for investigation of the fundamental understanding of activity and stability of IrOx nanoparticle catalysts using electrochemistry mass spectrometry and optical absorption spectroscopy to probe gas phase products and reaction intermediates. The results of these studies show an optically observable species on the nanoparticle catalysts that begins to occur approximately 100 mV less positive than oxygen onset, defined as the initial potential at which evolved oxygen can be directly observed either electrochemically or via the use of in-situ techniques, and tracks into the oxygen evolving region. Additionally, the differences observed between oxygen onset observed from mass spectrometry between amorphous and crystalline catalysts track with results observed from optical signals.Open Acces

    Security and Privacy for Modern Wireless Communication Systems

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    The aim of this reprint focuses on the latest protocol research, software/hardware development and implementation, and system architecture design in addressing emerging security and privacy issues for modern wireless communication networks. Relevant topics include, but are not limited to, the following: deep-learning-based security and privacy design; covert communications; information-theoretical foundations for advanced security and privacy techniques; lightweight cryptography for power constrained networks; physical layer key generation; prototypes and testbeds for security and privacy solutions; encryption and decryption algorithm for low-latency constrained networks; security protocols for modern wireless communication networks; network intrusion detection; physical layer design with security consideration; anonymity in data transmission; vulnerabilities in security and privacy in modern wireless communication networks; challenges of security and privacy in node–edge–cloud computation; security and privacy design for low-power wide-area IoT networks; security and privacy design for vehicle networks; security and privacy design for underwater communications networks

    Computational modeling of biological nanopores

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    Throughout our history, we, humans, have sought to better control and understand our environment. To this end, we have extended our natural senses with a host of sensors-tools that enable us to detect both the very large, such as the merging of two black holes at a distance of 1.3 billion light-years from Earth, and the very small, such as the identification of individual viral particles from a complex mixture. This dissertation is devoted to studying the physical mechanisms that govern a tiny, yet highly versatile sensor: the biological nanopore. Biological nanopores are protein molecules that form nanometer-sized apertures in lipid membranes. When an individual molecule passes through this aperture (i.e., "translocates"), the temporary disturbance of the ionic current caused by its passage reveals valuable information on its identity and properties. Despite this seemingly straightforward sensing principle, the complexity of the interactions between the nanopore and the translocating molecule implies that it is often very challenging to unambiguously link the changes in the ionic current with the precise physical phenomena that cause them. It is here that the computational methods employed in this dissertation have the potential to shine, as they are capable of modeling nearly all aspects of the sensing process with near atomistic precision. Beyond familiarizing the reader with the concepts and state-of-the-art of the nanopore field, the primary goals of this dissertation are fourfold: (1) Develop methodologies for accurate modeling of biological nanopores; (2) Investigate the equilibrium electrostatics of biological nanopores; (3) Elucidate the trapping behavior of a protein inside a biological nanopore; and (4) Mapping the transport properties of a biological nanopore. In the first results chapter of this thesis (Chapter 3), we used 3D equilibrium simulations [...]Comment: PhD thesis, 306 pages. Source code available at https://github.com/willemsk/phdthesis-tex

    Neurodevelopmental disorders:from genes to regulatory elements

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