Precise spatial and temporal control of cellular adhesion and signal transduction events are necessary for accurate animal development. Given the necessity for cell communication in carrying out processes like cell fate specification, growth, cell migration and differentiation, it is not surprising that signaling transduction pathways, such as EGFR, BMP, Notch, Wingless and Hippo, are intimately involved. All these pathways encompass a cascade of molecular events over which there is exquisite spatial and temporal control. A wide array of mechanisms, involving a diverse set of molecules, acts to provide this regulatory control. One such molecule implicated in the BMP signaling pathway in Drosophila development is Kek5, a Leucine rich repeat and Immunoglobulin domain (LIG) family member. Here I show that Kek5 modulates both BMP signaling and adherens junctions. For these functions, I further demonstrate that structural elements in both extracellular and intracellular region of Kek5 are critical, providing new insight into the LIG family and their roles in signaling pathways.

Transepithelial barriers represent important mechanisms by which epithelial cells delimit tissue compartments and maintain distinct extracellular environments. Such cellular barriers are key in regulating organ and tissue homeostasis and their dysregulation leads to a wide variety of pathologies. Novel tight junctions termed tricellular junctions (TCJs) appear to provide this barrier activity at the molecular level. Despite their proposed key role in barrier function, our understanding of these junctions is limited, with only a few molecules localized to tricellular junctions having been reported. Here we add to this understanding by identifying a LIG family member, Kek6, in Drosophila that represents the first example of a molecule uniquely excluded from TCJs. LIGs represent transmembrane molecules with Leucine-rich repeats and Immunoglobulin domains whose expression is often enriched in the developing nervous system. Data on Kek6 confirms this nervous system expression. Investigation into the mechanism which controls Kek6€™s unique exclusion from TCJs has proved that it is not solely mediated by the C-terminal intracellular PDZ domain-binding site. Although PDZ domain-binding sites of various proteins have been implicated as important for protein localization, it is thought that it is the extracellular domain of Kek6 that is the part of the protein which is responsible for its unique localization pattern. Shown here, it is believed that Kek6 participates in a stabilizing homophilic interaction which may support the hypothesis that the extracellular domain is required for localization. Kek6 expression in one cell is not sufficient for expression in the bicellular junctions. Adjacent cells must both express Kek6 in order for Kek6 to be stably localized to the bicellular junction. Studies on the potential relationship between Kek6 and Gliotactin, the Drosophila protein which localizes to TCJs, revealed that there is no direct relationship between these two proteins but does not eliminate the potential of unidentified shared interactors. Further investigation of Kek6 will allow for the elucidation of the role of Kek6 in TCJs which will help further develop the junction biology field. In addition to the information provided on Kek6, this study reports the first localization and functional knowledge of Kek3. Misexpression of Kek3 leads to cross vein defects and reduction/loss of bristles revealing that Kek3 may be a modulator of BMP signaling. Although family member Kek5 has been previously identified as a modulator of BMP signaling, the mechanism of this function is still under investigation but it is believed that Kek3 is acting through a different mechanism.

The development of Laser-Induced Thermal Grating Spectroscopy (LITGS) for diagnostics of combusting and non-combusting flows is described. The first use of LITGS to provide in situ calibration of 2-Dimensional temperature distributions generated using Two-Colour Planar Laser-Induced Fluorescence (TC-PLIF) is reported. Time-resolved measurements of temperature distributions in a firing GDI optical engine obtained by TC-PLIF were made during the compression stroke and calibrated to the absolute temperature scale by simultaneous LITGS measurements. The accuracy and precision of the temperatures derived from LITGS data are evaluated using alternative methods of data analysis - Fast Fourier Transform and Fitting to theoretical models of the experimental data. The relative merits of the two methods are examined for analysis of weak LITGS signals obtained under engine conditions of low pressure and high temperature. The combined TC-PLIF and LITGS system was demonstrated by performing repeated single-shot measurements for 1 in every 10 four-stroke cycles showing excellent correlation of the temperatures derived from both techniques. Direct measurement of the effect of 'charge cooling', of order 5 K, for operation with direct injection is reported. Inhomogeneous temperature distributions were observed during the compression stroke for fired operation with Port Fuel Injection (PFI) and also with Gasoline Direct Injection (GDI). The effects of varying the relative concentrations of toluene and iso-octane in the two-component fuel were investigated. Extension of the LITGS technique to multi-point measurements along a 1-D line is described. By recording signals from 4 points on separate detectors using a fibre-coupled photodiode array the limitations of Streak Cameras used previously for 1-D LITGS measurements were overcome. Demonstration of principle experiments are reported in which simultaneous 4-point measurements were made with 1 mm spatial resolution and a precision of 0.7 % in temperature gradients in gas flows and in boundary layers at surfaces.