Laboratories offering studentships for the 2018 summer school are listed below together with potential projects. On the registration form you will be asked to rank your top three in order of preference.
We encourage you to read recent papers from the labs which interest you before making your choices.
"Imaging the cytoskeleton of myometrial smooth cells (MSCs) under agonist stimulation"
(Joint with Anne Straube)
Uterine myometrial cells are a highly specialised type of smooth muscle. MSCs generate more force per cell than any other smooth muscle, are steroid hormone responsive, electrically active and undergo major hypertrophy during gestation. We have developed an immortalised cell line from late gestation human myometrium as a model to study the detail of these cellular dynamics. The summer project in collaboration with Anne Straube will use imaging to study the effect of agonist stimulation on the dynamics of the myometrial cell cytoskeleton (microtubules, actin, actinin).
"Quantitative modeling of cellular metabolism"
(Joint project with Pat Unwin)
Pat Unwin (Co-I) Metabolism consists of a myriad, interconnected biochemical reactions and underpins cellular physiology from microbes to human cells. It has been increasingly recognized that cells also display significant “metabolic overflows”, where several of the intracellular metabolites can be secreted (actively or passively) to the outside of the cell. This results in metabolic interactions among cells within tissues (or tumors) in higher organisms and within self-organizing spatial structures in microbial communities. There are currently no quantitative, i.e. data-driven models for metabolic secretions under different environmental conditions. In this project, we will quantify metabolic secretions in select model microorganism(s), as well as key micro-environment parameters such as oxygen, pH, and redox potential. This data will inform a quantitative, mathematical model of the central metabolism, including glycolysis and the Krebs cycle, where many of the metabolic overflows originate from. Current models of such systems solely focus on internal dynamics and usually ignore effects arising from thermodynamic and environmental limitations.
 Examples of metabolic interactions in microbial communities: https://www.ncbi.nlm.nih.gov/pubmed/27258948;https://www.biorxiv.org/content/early/2017/09/28/195339
 Example of metabolic interactions in cancer; https://www.ncbi.nlm.nih.gov/pubmed/24218566
 Example of mathematical modeling of the TCA cycle; https://www.ncbi.nlm.nih.gov/pubmed/27329289; https://www.ncbi.nlm.nih.gov/pubmed/25089525
"Watch the tick-tock of the clock: Real-time recording of cellular circadian clocks with substrate free bioluminescence reporters"
Biological circadian clocks have evolved in practically all living organisms and enable them to anticipate regular changes in the environment. They orchestrate a significant portion of cellular and organismal physiology. Disruption of the clock mechanism can lead to disease or worsen the progression of diseases. Using bioluminescence reporter constructs that translate circadian gene expression to light production, which can be easily monitored, has allowed real-time observation of cellular clocks. However, widely used firefly luciferases always have to be fed a substrate to produce light. Here, the goal is to develop a substrate free circadian reporter system based on a bacterial luciferase operon.
“Designing a millifluidic gut model”
Developing a robust in vitro model of the human gut is essential for probing biological interactions of the anaerobic bacteria with gut cells. This project aims to redesign and develop further a static dual environment cellular gut model by introducing controlled flow.
“Calcium dynamics during bacterial cellular differentiation into dormant spores”
Calcium is a universal signaling molecule involved in many cellular functions. Recent studies revealed calcium also plays important roles in bacterial cells, however, the molecular dynamics of calcium is still poorly understood. This project aims to gain insights to the calcium dynamics during bacterial cellular differentiation into spores. Under stress conditions, Bacillus subtilis can develop into dormant spores which are highly resilient against almost all kinds of chemical and physical stresses (e.g. Heat, UV, antibiotics and lysozymes). It has been known that B subtilis accumulate high level of calcium during spore formation in an energy dependent manner. However, how cells accumulate an extreme level of calcium during sporulation is largely unknown. Using the fluorescent calcium indicator, GCaMP6, we have measured the calcium dynamics at the single cell level throughout the spore formation. Our preliminary data suggested the calcium uptake may be regulated by a sporulation sigma factor. Our single-cell measurements also suggested the timing of the calcium uptake may be different from the commonly accepted view based on population-level measurements. To further investigate the above findings, this project will use the GCaMP6 to monitor the calcium dynamics at single cell level, using various genetic mutant strains.
“Defining the role of regulatory RNA in bacterial stress responses”
To survive bacteria must respond correctly to the environment in which they find themselves, whether this is during infection in the case of a pathogen, in response to changes in nutritional levels, or in response to chemical or physical stress (such as antibiotics or change in temperature). Complex regulatory systems are present to ensure that genes needed to survive in a particular condition are correctly switched on or off. It was recently discovered that regulatory RNAs play important roles in these processes, which is the focus of my research. Many regulatory RNAs function by interacting with other RNAs over non-consecutive nucleotides which makes predicting the role of regulatory RNAs very challenging. To tackle this we have developed an RNA-seq method to define all RNA-RNA interactions that are taking place in any bacterial cell at any one time. This allows us to make informed decisions on the correct way to study the function of any particular RNA at the molecular level, for example using GFP to report on the activity of particular targets of the RNA. Understanding fundamental aspects of bacterial cell biology is important for defining new targets for antimicrobials and for optimizing bacterial cell factories.
"Modulation of stochastic protein expression profiles"
Knowledge about the genomic landscapes of various types of cancers has increased significantly in recent years. However, comparatively little is known about the initial causes of various aberrations, such as mutations and chromosomal structural changes. Evidence is accumulating that stochastic events such as replication errors play a significant role . This is probably not surprising given that cells are in general ‘noisy’ systems, with great variations in protein and mRNA expression levels among otherwise identical cells [2, 3]. Yet, data that relate natural stochastic expression variation of various factors to incidence of aberrations are scarce  or address the issue indirectly . To investigate the importance of stochastic expression properties of several cancer-associated factors, we want to perturb the expression profile of a selected factor in ways that keeps the mean level of protein expression across a cell population the same, while altering the distribution of expression levels among individual cells, in particular their variance/standard deviations. This will permit to specifically investigate the importance of a factor’s expression noise in the accumulation of aberrations independently from its overall expression level. The work will be based on transient transfection of an expression vector in a mammalian cell line and will include modification of the vector by codon optimization, addition of degron tags, and similar techniques .
1. Tomasetti, C., et al., Science, 2017. 355(6331): p. 1330. 2. Balazsi, G., et al., Cell, 2011. 144(6): p. 910. 3. Raj, A., et al., Cell, 2008. 135(2): p. 216. 4. Uphoff, S., et al., Science, 2016. 351(6277): p. 1094. 5. Paek, A.L., et al., Cell, 2016. 165(3): p. 631. 6. Pilpel, Y., Methods Mol Biol, 2011. 759: p. 407.
"Chromatin in motion"
Eukaryotic chromosomes are one of the largest and most complex structures occuring in nature. My research interests lie in the structure-function relationship of human chromosomes, particularly in the development of tools that allow us to observe chromatin dynamics in living cells. We have recently developed a technique whereby we can label newly replicated chromatin through pulse-labelling of the histone component, allowing us to observe the dynamics of chromatin domains in living cells over multiple generations. The summer project will involve the application of this approach in cultured mammalian cells, and give the student a chance to learn techniques in live cell imaging, molecular cloning, image analysis and mammalian cell culture.
Three potential summer projects are available in my lab:
1. Making DNA constructs for genome editing in human cells to label the cell's cytoskeleton with fluorescent protein markers.
2. Imaging cells that are infected with a virus and study the steps in intracellular transport of the virus from the assembly of the capsid in the nucleus to the plasma membrane.
3. Studying the dynamics of microtubules formed from purified proteins outside of the cell.
“Measuring and modelling protein movement in human cells”
Cells are crowded environments. Proteins must be recruited quickly to specific places in order for cells to carry out important function. For small proteins, simple diffusion might explain how they can move to a target site, but there are many obstacles in the way. For larger proteins, their movement is predicted to be extremely limited, yet they can apparently move large distances in the cell to find their target rapidly. How this works is unclear. In this project, you will measure the rates of protein translocation from A to B for a variety of differently sized proteins using live cell microscopy. Analysis of these images will allow us to see what rules govern protein movements in cells. Finally, we will then model these movements using known physical parameters in a simulated cell environment.
I can offer three potential projects in my lab:
1. “Can molecular motors drive microtubule self-joining?”
A little-studied aspect of microtubule dynamics is microtubule end-to-end joining (ligation). This process, which allows short microtubules to suddenly get much longer, may be important in cells, in situations where microtubule become crowded together. You will visualise self-joining by fluorescence microscopy and investigate whether microtubule molecular motors can influence self-joining.
2. “Motor-driven driven microtubule motility in lifelike solutions.”
In almost all experiments on purified motors and microtubules, we use an assay buffer that only slightly resembles cytoplasm. In this project you will develop a buffer that is more cytoplasm-like, and investigate whether it influences the stepping of molecular motors along their microtubule tracks.
3. “Imaging the response of microtubule dynamics to drugs using microfluidics.”
Recent work suggests that medically-important microtubule-stabilising drugs have unexpected effects on microtubule structure and dynamics when the drugs are present only at low concentrations. You will look into this problem using video microscopy of dynamic microtubules in microfluidics circuitry that allows drug concentrations to be varied precisely in real time.
“Stopping DNA synthesis: analysis of the effect of a hyperactive version of Mec1 on DNA replication.”
DNA is the repository of cellular genetic information. Each time a cell duplicates, all of this information must be fully and faithfully copied. Cells have evolved a specialised machine to conduct this complicated task (the replisome). They have also evolved sensors that monitor defects that may occur during DNA replication. When activated, these sensors trigger a complex and far-reaching signalling response that controls cell cycle progression, protein production, and the replisome. This response is called the S phase checkpoint. How the S phase checkpoint controls the replisome is, however, still poorly understood. One possibility is that the checkpoint actively regulates the replisome and stops DNA synthesis. Direct testing of this hypothesis is difficult, though, because the chemicals used to activate the S phase checkpoint also intrinsically interfere with DNA replication. The aim of this study is to explore whether the S phase checkpoint directly regulates the rate of DNA synthesis. To test this, we aim to generate by mutagenesis a hyperactive version of the key checkpoint sensor Mec1. This will allow us to analyse, in the absence of DNA damage, whether Mec1 directly controls the rate of DNA synthesis.
"Evaluating the S. cerevisiae potential as a model for fungal pathogenic traits"
Superficial and invasive fungal infections are caused by commensal or environmental fungi bearing pathogenic traits making them able to elude the host defenses and instaurate infections. Several phenotypes are known to be related to fungal pathogenicity, but the mechanism of instauration is known for only a few of them. S. cerevisiae has been used as a model for several purposes, but the common consideration of this yeast as a GRAS (Generally Recognized As Safe) microorganism and the usage of a single laboratory strain largely limited its exploitation for the dissection of the molecular mechanisms lying at the basis of fungal pathogenic traits. The project aims at the exploration and quantification of the variance of pathogenic traits in a pool of whole-genome sequenced S. cerevisiae strains to evaluate the potential of this yeast as a model with the final intent to link the strains’ genetics to the pathogenic traits variability.
Microtubule bundle structures such as the central spindle play crucial roles in animal cytokinesis. We have recently discovered that the direct interaction between the two different types of microtubule bundling proteins, PRC1 and centralspindlin, is crucial for stable maintenance of the central spindle when it is under the mechanical tension . Mutant C. elegans embryos defective for this interaction shows the rupture of the central spindle during anaphase. I propose the following two projects:
Project 1. "In vivo dynamics of the central spindle"
A model for the spindle elongation based on coupled sliding and polymerisation of microtubules predicts that the promotion of microtubule polymerisation should suppress this ‘split spindle’ phenotype. We will test this hypothesis by observing the effect of RNAi-mediated depletion of various regulators of microtubule dynamics on this phenotype using advanced live microscopy.
Project 2. "In vitro characterisation of a centralspindlin mutant"
Through a genetic screen, we found that a mutation in the kinesin motor subunit of centralspindlin can suppress the lethality of a mutant defective for the PRC1-centralspindlin interaction. In this project, the mutant and wild-type versions of the motor subunit constructs will be purified from recombinant bacteria and their microtubule-motor activities will be examined.
 Lee, K.-Y., Esmaeili, B., Zealley, B., and Mishima, M. (2015). Nat Commun 6, 7290 (open acess).
“Memory and aging”
Recent advances have shown that age-related diseases share common causes and thus can be delayed or reversed through common cures. We study small, short-lived nematodes to understand how manipulations of the environment, such as diet, can extend their health span. This project will involve the design and implementation of microfluidics systems to assay memory formation in the nematode C. elegans as proxy for health. Microfluidics offer advantages compared to traditional methods because they allow the precise control of concentration of molecules, and confinement, monitoring and stimulation of nematodes. The interdisciplinary research will take place in the School of Life Sciences and the Department of Physics at the University of Warwick under supervision of Dr. Andre Pires da Silva and Dr. Vasily Kantsler.
"Novel Signalling Pathways in Brown Adipocytes"
Brown adipocytes serve a unique role in burning fat to generate heat. Therefore, they represent a promising therapeutic target for promoting weight loss. This project will investigate receptor-mediated signalling pathways in brown adipocytes and how they control cellular bioenergetics.
"Synthetic Biological Reconstruction of a vertebrate signalling pathway"
Work will involve generation of a synthetic signalling pathway using unnatural amino acids incorporated into key signalling proteins through genetic code expansion. We will make proteins with a 21st amino acid at specific locations! Through these analysis, a signalling pathway will be constructed from first principles. In particular, we will investigate the role of protein phosphorylation of the transcription factor Smad2 in signalling and the regulation of phosphorylation by receptor serene kinase – ligand interactions.
"Imaging morphogen dynamics in vivo"
(Joint project with Till Bretschneider)
Animals develop from a single cell to form multicellular organisms. Signals called “morphogens” instruct animal cells how to behave during development and differentiation. Morphogens move across cells and tissues to form gradients of the signal. Cells then respond in different ways depending on how much of the signal they receive. We are studying a morphogen called "Nodal" in zebrafish. The project will use live-imaging and computational analysis of imaging data to determine the kinetics of Nodal morphogens in vivo.
Antifreeze proteins (AFPs) have a vast array of potential industrial, biotechnological and pharmacological applications. They are thought to bind to small ice crystals and in doing so inhibit their growth into larger crystals which may have deleterious effects on biological entities. The mechanism involving this mode of action, however, isn’t entirely clear. This summer project will involve the heterologous expression of one or more of these AFPs in Escherichia coli. Subsequently, their antifreeze activity in cells as well as their interaction with ice crystals will be investigated through biophysical methods and biological assays.