The development and use of fluorescence microscopy technologies has allowed significant breakthroughs to be made in our understanding of the fundamental processes within a cell. This includes the ability to observe differences in the behaviour of molecules depending upon their cellular location. One break-through which has facilitated this has been the development of Fluorescence Lifetime Fluorescence Imaging Microscopy (FLIM), where specific lifetime determination of any fluorophore tagged molecule within a cell can be determined. Research within the Mulvihill and Warren labs at the University of Kent have highlighted the importance of mechanisms for spatially regulating protein stability within the bacterial and fungal cell. Compartmentalization of metabolic activities represents an important tool by which defined microenvironments can be created for specific metabolic functions. This provides challenges for bacteria, which lack membrane bound organelles, however some overcome this by making specialized proteinaceous metabolic compartments called bacterial micro-compartments (BMCs) or metabolosomes. The Warren and Mulvihill labs have recently reported (J. Biol, Chem. 283: 14366-75; Mol. Cell 38: 305-15) that using synthetic biology techniques not only could the shell of an empty BMC can be produced within E. coli cells but proteins of interest can be targeted to the empty BMC, thus providing a controlled microenvironment within the cell to optimize the stability of recombinant proteins. Although this finding is likely to have a significant impact for both biopharmaceutical and biotechnology applications, its full potential in terms of controlling the environment within the BMC and subsequent stabilisation of target proteins within them have yet to be explored. Coincident work in the Mulvihill lab using the fission yeast has recently uncovered a novel mechanism in which a class V myosin modulates the spatial coordination of proteolysis of the S. pombe CLIP-170 homologue (J.Cell Sci. 122: 3862-72.). The myosin works in concert with a ubiquitin receptor to enhance Clip170 removal from the plus end of growing microtubules at the cell tips and target it for degradation, and thus regulate microtubule dynamics. However the sites of Clip170 degradation, stability and high turn over remain unresolved, as do the turnovers of other cytoskeletal regulators. This project sets out to develop an imaging system to facilitate Fluorescence Lifetime Fluorescence Imaging Microscopy (FLIM) to determine differences in the exponential decay rate of fluorescence of targeted molecules, depending upon their cellular location in both bacterial and fission yeast systems, and will allow the PhD student to determine (i) the protection synthetic bacterial micro-compartments allow molecules that have been targeted to their interior; and (ii) spatial differences in the turnover of regulators of eukaryote cytoskeleton dynamics, which provides a means to control cell polarity and growth. These research questions coincide exactly with current development projects within Cairn Research, a leading developer of LED illumination technology for biological applications, who have recently developed a proprietary FLIM system to be used in conjunction with their world leading LED light sources. These state-of-the art LEDs provide a stable light source in which intensity and intensity modulation can be exquisitely controlled. The facility to 'gate' the light source directly from the camera and thus only expose the specimen for extremely short (msec) controlled periods is crucial for FLIM analyses and make it an optimum light source for this application. This will have the potential to provide significant cost savings over conventional laser based systems. Therefore this synergy of research interests and close proximity in geographical locations provide an excellent opportunity for both researchers at Cairn and UKC for developing and optimising this FLIM system.

One in six couples experience difficulties conceiving, with male factors present in approximately half of all cases. The major factor affecting natural conception is probably ability of sperm to reach the egg, with only 8-20 sperm from over 200 million ever reaching the egg even in fertile couples. As yet there is no successful drug treatment targeted to improve male fertility. The best hope for rational therapy will derive from a detailed understanding of sperm energetics and this can only be achieved through detailed analysis of the sperm's swimming dynamics. This programme will provide detailed 3-dimensional movies of swimming sperm, offering new insights into sperm dynamics that will enable next generation individualised diagnostics and the rational screening of new drug treatments.

One in six couples experience difficulties conceiving, with male factors present in approximately half of all cases. The major factor affecting natural conception is probably ability of sperm to reach the egg, with only 8-20 sperm from over 200 million ever reaching the egg even in fertile couples. As yet there is no successful drug treatment targeted to improve male fertility. The best hope for rational therapy will derive from a detailed understanding of sperm energetics and this can only be achieved through detailed analysis of the sperm's swimming dynamics. This programme will provide detailed 3-dimensional movies of swimming sperm, offering new insights into sperm dynamics that will enable next generation individualised diagnostics and the rational screening of new drug treatments.

The development and use of fluorescence microscopy technologies has allowed significant breakthroughs to be made in our understanding of the fundamental processes within a cell. This includes the ability to observe the actin and microtubule cytoskeletons, which are the extremely dynamic polymer based intracellular structures responsible for the internal organisation within a cell and allow the cell to respond rapidly to changes in both the intra- and extra- cellular environments. The actin cytoskeleton plays an important role in a plethora of conserved cellular processes within cells. These include the transport of molecules to distinct cellular locations. This is facilitated by myosin motor proteins, which move along actin filaments to deliver cargoes to specific cellular locations. The speed at which the myosins move within the cell (up to 10 um/sec) can make visualising their movements in vivo extremely challenging. Over recent years this lab has been using the fission yeast model system to study the motility and function of the class V myosins, Myo52. This has led to the development of strains and techniques to visualise this myosin's movement in vivo, and the identification of a number of cargoes for these motor proteins (Cell Motil Cytoskeleton 2006 63: 149; J Cell Sci 2007 120: 4093; J. Cell Sci. 2009 122: In press; J. Cell Sci. 2009 122: In press.). However, limitations in current off the shelf bio-imaging equipment have made it difficult / impossible to examine whether the speed of the motor is affected by interactions with: (a) different cargoes (e.g. through protein folding / regulating post-translational modifications / mass increase); or (b) different types of actin polymer, where the presence of different actin binding proteins may regulate myosin movement. This hypothesis is supported by our finding that Myo52 movements fall into at least two populations. This project sets out to (i) Develop an imaging system to facilitate automated Fluorescence Resonance Energy Transfer (FRET) based system biology screens to define proteins which interact with either of the fission yeast class V myosins, and (ii) To simultaneously track the position and dynamic behaviour of the actin track, the myosin motor and its cargo, to examine how their interactions affect motility and whether cargo and track affect the velocity of these molecular motors in vivo. (i) The FRET based screen will be developed using known interacting and non-interacting proteins to optimise screen conditions. The student will optimise the light path and write software scripts to allow automated screening using multiwell plates. Then using a fission yeast YFP fusion expression vector library, the student will look for FRET interactions with Myo52-CFP, in a system biology based fluorescence screen. This screen can then be applied to define interacting proteins for each cytoskeleton component studied within this lab. Each positive result will subsequently be confirmed by yeast 2-hybrid and biochemical techniques. (ii) We will develop a bioimaging system capable of following movements throughout the entire cell using 3 wavelengths (track motor and cargo) in the sub-second time-scale. Previous work in this lab has defined specific protein cargoes of Myo52. The student will generate yeast strains in which Myo52, actin filaments and cargoes are tagged with different combinations of CFP, GFP and mCherry fluorophores, and define which one allows optimum visualisation in combination with optimum functionality. This will introduce the student to the rigours of careful controls and careful experimental procedures of a repetitive nature. Using proprietary technologies developed by Cairn, the student will develop the imaging system to allow simultaneous observation of the three wavelengths or multiple z layers. The student will highlight which aspects of the technology can be improved on, and make developments which can be applied to future imaging systems.

Infertility, not being able to conceive after a year of trying for a baby, affects around one in six couples. Problems with sperm - for example low numbers of rapid swimming sperm, or poorly-formed sperm (which may have damaged DNA) contribute in around half of all cases. Therapies such as IVF, or ICSI (injection of sperm into an egg) are used to treat sperm-related problems, however they are ineffective, are very expensive and put physical and emotional strain on the couple, particularly the woman. Worryingly, using sperm with damaged DNA may contribute to miscarriage or health problems in any resulting children. Some patients would be better off continuing to try for a baby naturally, but with some lifestyle changes (stopping smoking, improving diet), or with a less invasive treatment (insemination into the womb). Other patients should be quickly moved to IVF, some will only conceive through ICSI. The difficulty with treatment decisions is that methods to determine the type and severity of sperm problems are imprecise. The main methods are manually 'counting' swimming sperm, and drying them out on a slide to examine their shape. This does not take advantage of the huge leaps in computer and camera technology made in recent years - indeed even 1980s 'Computer-Aided Semen Analysis' methods are not generally used in clinics (partly because of their unsatisfactory accuracy). Think of the technology in a typical smartphone - a high definition (possibly rapid framerate) camera, pattern recognition, and high volume data processing/storage - this is the type of 21st Century technology that needs to be brought into the fertility clinic. We will develop a new way to examine sperm, using both rapid digital camera imaging, and computer-based pattern recognition. The aim will be to be able to automatically, accurately and repeatably examine a semen sample, collecting simultaneous data on how cells swim, and what their shape is like. The target of this technique will be to look for the 'special' few cells that have the right shape, and can swim well - so that in natural fertilisation they would be able to travel through the cervix, womb and fallopian tubes and fertilise the egg. There will be a number of practical issues that will need to be solved. For example, do we need to make the cells fluorescent so we can see their shape better, or can we achieve our aims with a 'standard' type of microscopy? Can we work with samples at any concentration, or do we need to dilute them to recognise the cells properly? Should we use a 'micro-fluidic' chamber to separate out the swimming cells first - and should we use a high viscosity ('sticky') fluid that better represents the physical challenge sperm face in the female reproductive tract? A key question will be how to convert the large volume of information we can measure into information that doctors and patients can make use of. We will apply a type of machine learning based on prototypes, representations of typical types of patients based on the many 'features' we can extract from rapid sperm videos. These prototypes will be progressively modified as more patients come through the system, making the model more accurate. In the long term the possibility of integrating the large volume of data through a model we can train will lead to a very powerful way to bring together clinical information nationally or even internationally. As the system makes its way into real application, patients will receive the right treatment more quickly, saving resources, patients will have a less difficult experience of fertility treatment and will achieve success more quickly. A longer-term benefit will be by helping clinical research and toxicology - the system will provide researchers with a powerful method to test new advances in fertility treatment, for example drugs, lifestyle changes, and they will also be able to check for unintended sperm-toxic effects of other chemicals.