Unravelling the Cell Adhesion Defect in Meckel-Gruber Syndrome

Meckel-Gruber syndrome (MKS) is a universally lethal heritable human disease characterised by CNS malformations, cystic kidney, polydactyly, and liver fibrosis. MKS is classed as one of the ciliopathies due to its association with dysfunctional primary cilia, signalling organelles found on most cells in the human body. Some of the symptoms of MKS can be explained as a consequence of disrupted developmental signalling through the primary cilium, other defects are harder to explain, and evidence now exists for non-ciliary influences on ciliopathies. The nature of these influences, and the implications they may have for our understanding of ciliary function and the aetiology of MKS, remain unclear. In this thesis, defects in cell-extracellular matrix (ECM) interaction in MKS are investigated to determine whether MKS proteins have a role in this process, and if so, whether this role may be involved in MKS pathology. A combination of transcriptomic, proteomic, and cell imaging approaches are used to demonstrate that MKS patient cells produce a defective extracellular matrix, and that the MKS protein TMEM67 is present at the cell surface at sites of cell-ECM interaction. It is shown that the full-length TMEM67 protein is required for correct ECM morphology, and it is further shown that the abnormal extracellular matrix morphology in MKS cells underlies other defects, including failure to build cilia and alterations to the actin cytoskeleton. This represents the first set of causal relationships identified between the cellular defects in this complex disease. It is further shown that treatment with developmental signalling pathway antagonists can rescue these defects, potentially revealing a new avenue of therapeutic intervention for MKS. Finally, possible upstream defects are investigated that might underlie the ECM defect, including alterations to cell spreading behaviour and cell deformation resistance

Is the Pale Blue Dot unique? Optimized photometric bands for identifying Earth-like exoplanets

The next generation of ground and space-based telescopes will image habitable planets around nearby stars. A growing literature describes how to characterize such planets with spectroscopy, but less consideration has been given to the usefulness of planet colors. Here, we investigate whether potentially Earth-like exoplanets could be identified using UV-visible-to-NIR wavelength broadband photometry (350-1000 nm). Specifically, we calculate optimal photometric bins for identifying an exo-Earth and distinguishing it from uninhabitable planets including both Solar System objects and model exoplanets. The color of some hypothetical exoplanets - particularly icy terrestrial worlds with thick atmospheres - is similar to Earth's because of Rayleigh scattering in the blue region of the spectrum. Nevertheless, subtle features in Earth's reflectance spectrum appear to be unique. In particular, Earth's reflectance spectrum has a 'U-shape' unlike all our hypothetical, uninhabitable planets. This shape is partly biogenic because O2-rich, oxidizing air is transparent to sunlight, allowing prominent Rayleigh scattering, while ozone absorbs visible light, creating the bottom of the 'U'. Whether such uniqueness has practical utility depends on observational noise. If observations are photon limited or dominated by astrophysical sources (zodiacal light or imperfect starlight suppression), then the use of broadband visible wavelength photometry to identify Earth twins has little practical advantage over obtaining detailed spectra. However, if observations are dominated by dark current then optimized photometry could greatly assist preliminary characterization. We also calculate the optimal photometric bins for identifying extrasolar Archean Earths, and find that the Archean Earth is more difficult to unambiguously identify than a modern Earth twin.Comment: 10 figures, 38 page

Live cell imaging of lipid droplet distribution and motility in the filamentous fungus Ustilago maydis

Lipid droplets (LDs) are organelles specialised for lipid metabolism and storage, found across the domains of life. They are dynamic in number and size, actively transported, and have diverse functions, many of which have only recently been identified. Despite this, they remain less well-characterised than many other organelles. While the motility of LDs has been noted in filamentous fungi, no study has yet investigated its mechanism. In this study, several techniques were established for visualisation of LDs in live cells of the dimorphic fungus Ustilago maydis. This species is a prominent pathogen of maize (Zea mays) and an established model organism for intracellular trafficking. Distribution and motility patterns of LDs were investigated quantitatively in U. maydis cells under varying growth conditions, including during plant infection. Active transport of LDs was found to be microtubule-dependent, and dependent on specific motor proteins and organelle interactions

Akt promotes Endocardial-Mesenchyme Transition

Endothelial to mesenchyme transition (EndMT) can be observed during the formation of endocardial cushions from the endocardium, the endothelial lining of the atrioventricular canal (AVC), of the developing heart at embryonic day 9.5 (E9.5). Many regulators of the process have been identified; however, the mechanisms driving the initial commitment decision of endothelial cells to EndMT have been difficult to separate from processes required for mesenchymal proliferation and migration. We have several lines of evidence that suggest a central role for Akt signaling in committing endothelial cells to enter EndMT. Akt1 mRNA was restricted to the endocardium of endocardial cushions while they were forming. The PI3K/Akt signaling pathway is necessary for mesenchyme outgrowth, as sprouting was inhibited in AVC explant cultures treated with the PI3K inhibitor LY294002. Furthermore, endothelial marker, VE-cadherin, was downregulated and mesenchyme markers, N-cadherin and Snail, were induced in response to expression of a constitutively active form of Akt1 (myrAkt1) in endothelial cells. Finally, we isolated the function of Akt1 signaling in the commitment to the transition using a transgenic model where myrAkt1 was pulsed only in endocardial cells and turned off after EndMT initiation. In this way, we determined that increased Akt signaling in the endocardium drives EndMT and discounted its other functions in cushion mesenchymal cells

The pale orange dot : the spectrum and habitability of hazy Archean Earth

Recognizing whether a planet can support life is a primary goal of future exoplanet spectral characterization missions, but past research on habitability assessment has largely ignored the vastly different conditions that have existed in our planet's long habitable history. This study presents simulations of a habitable yet dramatically different phase of Earth's history, when the atmosphere contained a Titan-like, organic-rich haze. Prior work has claimed a haze-rich Archean Earth (3.8–2.5 billion years ago) would be frozen due to the haze's cooling effects. However, no previous studies have self-consistently taken into account climate, photochemistry, and fractal hazes. Here, we demonstrate using coupled climate-photochemical-microphysical simulations that hazes can cool the planet's surface by about 20 K, but habitable conditions with liquid surface water could be maintained with a relatively thick haze layer (τ ∼ 5 at 200 nm) even with the fainter young Sun. We find that optically thicker hazes are self-limiting due to their self-shielding properties, preventing catastrophic cooling of the planet. Hazes may even enhance planetary habitability through UV shielding, reducing surface UV flux by about 97% compared to a haze-free planet and potentially allowing survival of land-based organisms 2.7–2.6 billion years ago. The broad UV absorption signature produced by this haze may be visible across interstellar distances, allowing characterization of similar hazy exoplanets. The haze in Archean Earth's atmosphere was strongly dependent on biologically produced methane, and we propose that hydrocarbon haze may be a novel type of spectral biosignature on planets with substantial levels of CO2. Hazy Archean Earth is the most alien world for which we have geochemical constraints on environmental conditions, providing a useful analogue for similar habitable, anoxic exoplanets.Publisher PDFPeer reviewe

Neutron Thermal Cross Sections, Westcott Factors, Resonance Integrals, Maxwellian Averaged Cross Sections and Astrophysical Reaction Rates Calculated from Major Evaluated Data Libraries

We present calculations of neutron thermal cross sections, Westcott factors, resonance integrals, Maxwellian-averaged cross sections and astrophysical reaction rates for 843 ENDF materials using data from the major evaluated nuclear libraries and European activation file. Extensive analysis of newly-evaluated neutron reaction cross sections, neutron covariances, and improvements in data processing techniques motivated us to calculate nuclear industry and neutron physics quantities, produce s-process Maxwellian-averaged cross sections and astrophysical reaction rates, systematically calculate uncertainties, and provide additional insights on currently available neutron-induced reaction data. Nuclear reaction calculations are discussed and new results are presented.Comment: 145 pages, 15 figures, 19 table