Identification of regulators and effectors of RhoGTPase signalling in corneal epithelial cells

Epithelial cells adhere to each other and are connected via a series of junctions. Tight junctions (TJs) are a specific type of junction consisting of heteromeric protein complexes that are linked to the actin cytoskeleton and are important in regulating paracellular permeability and cell polarity. RhoGTPases are small molecular switch proteins that are important regulators of the cytoskeleton and modulators of gene expression. RhoGTPases have thus been identified as being major signalling components associated with TJs. However little is known about how RhoGTPases are regulated to control junction formation and gene expression in corneal epithelial cells. I used a siRNA screening approach combined with functional assays to identify components of RhoGTPase signalling that affect the assembly of junctions and gene expression in Human corneal epithelial cells (HCE). I identified and validated several candidates that regulate junction assembly. One of these candidates was p114RhoGEF, a novel TJ localised guanine nucleotide exchange factor (GEF) important for the assembly of functional TJs. p114RhoGEF is a widely expressed and I discovered its depletion effects junction formation and morphogenesis in three dimensional culture systems in different epithelial cell types. p114RhoGEF is required for activation of RhoA at cell-cell junctions and junctional actinomyosin activity, p114RhoGEF is present in a complex containing Myosin II-A, the RhoA effector Rock II and the junctional adaptor protein Cingulin; indicating p114RhoGEF is a component of a junction associated RhoA-signalling module. p114RhoGEF, thus regulates spatial activation of RhoA at cell-cell junctions and organisation of the junctional cytoskeleton. p114RhoGEF may also have a role in cell migration, as depletion in HCE cells, caused cells to migrate at a slower rate during wound healing assays. I have also started to explore the function of a putative p114RhoGEF ortholog, cg10188 in Drosophila melanogaster. Preliminary experiments have identified cg10188 to be important in larval development

Tissue biochemical diversity of 20 gooseberry cultivars and the effect of ethylene supplementation on postharvest life

The European gooseberry (Ribes uva-crispa) is still an understudied crop with limited data available on its biochemical profile and postharvest life. A variety of polyphenols were detected in the skin and flesh of 20 gooseberry cvs, representing mainly flavonol glycosides, anthocyanins and flavan-3-ols. In contrast, gooseberry seeds were for the first time characterised by the presence of considerable amounts of hydroxycinnamic acid glycosides tentatively identified by UPLC-QToF/MS. All cvs examined represented a good source of vitamin C while being low in sugar. Furthermore, the postharvest stability of bioactives was explored by supplementation of exogenous ethylene in air at 5 °C. Results suggest a low sensitivity of gooseberries to ethylene. The overall quality of gooseberries remained stable over two weeks, showing potential for extended bioactive life

Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation

Epithelial cells develop morphologically characteristic apical domains that are bordered by tight junctions, the apical–lateral border. Cdc42 and its effector complex Par6–atypical protein kinase c (aPKC) regulate multiple steps during epithelial differentiation, but the mechanisms that mediate process-specific activation of Cdc42 to drive apical morphogenesis and activate the transition from junction formation to apical differentiation are poorly understood. Using a small interfering RNA screen, we identify Dbl3 as a guanine nucleotide exchange factor that is recruited by ezrin to the apical membrane, that is enriched at a marginal zone apical to tight junctions, and that drives spatially restricted Cdc42 activation, promoting apical differentiation. Dbl3 depletion did not affect junction formation but did affect epithelial morphogenesis and brush border formation. Conversely, expression of active Dbl3 drove process-specific activation of the Par6–aPKC pathway, stimulating the transition from junction formation to apical differentiation and domain expansion, as well as the positioning of tight junctions. Thus, Dbl3 drives Cdc42 signaling at the apical margin to regulate morphogenesis, apical–lateral border positioning, and apical differentiation

Blob sizes and velocities in the Alcator C-Mod scrape-off layer

A new blob-tracking algorithm for the GPI diagnostic installed in the outboard-midplane of Alcator C-Mod is developed. I t tracks large-amplitude fluctuations propagating through the scrape-off layer and calculates blob sizes and velocities. We compare the results of this method to a blob velocity scaling from a simple blob-model for sheath-connected blobs. We further present initial results from a fully three-dimensional blob model that features plasma resistivity as a free parameter

Micro-fabricated caesium vapour cell with 5mm optical path length

Micro-fabricated vapour cells have applications in a number of emerging quantum technology based devices including miniaturized atomic magnetometers, atomic clocks and frequency references for laser systems. Increasing the cell optical path length (OPL) and smallest cell dimension is normally desirable to increase the signal to noise ratio (SNR) and minimize the de-polarization rate due to collisions between atomic or molecular species and the cell walls. This paper presents a fully wafer-level scalable fabrication process to manufacture vapour cells with dimensions approaching those of glass-blown cells. The fabrication process is described and spectroscopic measurements (optical absorption and magnetic resonance) are reported. A magnetic resonance linewidth of 350 Hz is demonstrated, this is the smallest linewidth reported to date for a micro-fabricated vapour cell

Fast imaging of filaments in the X-point region of Alcator C-Mod

A rich variety of field-aligned fluctuations has been revealed using fast imaging of Dαemission from Alcator C-Mod's lower X-point region. Field-aligned filamentary fluctuations are observed along the inner divertor leg, within the Private-Flux-Zone (PFZ), in the Scrape-Off Layer (SOL) outside the outer divertor leg, and, under some conditions, at or above the X-point. The locations and dynamics of the filaments in these regions are strikingly complex in C-Mod. Changes in the filaments’ generation appear to be ordered by plasma density and magnetic configuration. Filaments are not observed for plasmas with n/nGreenwald≲ 0.12 nor are they observed in Upper Single Null configurations. In a Lower Single Null with 0.12 ≲ n/nGreenwald ≲ 0.45 and Bx∇B directed down, filaments typically move up the inner divertor leg toward the X-point. Reversing the field direction results in the appearance of filaments outside of the outer divertor leg. With the divertor targets “detached”, filaments inside the LCFS are seen. These studies were motivated by observations of filaments in the X-point and PFZ regions in MAST, and comparisons with those observations are made. Keywords: Alcator C-Mod; Turbulence; Divertor; X-point; Filament