Project offered for Ker Memorial PhD Studentship in Infectious Diseases at the University of Edinburgh
Trypanosomatid parasites cause devastating diseases in humans and their livestock, and these diseases predominantly affect low- and middle-income countries. A characteristic feature of these single-cellular organisms is their extraordinarily massive and complex mitochondrial DNA, also named kinetoplast DNA (kDNA) after the structure it is organised in.
Kinetoplast gene expression is critical for survival of these parasites, and interference with this process is an important part of the mode-of-action of some existing anti-trypanosomatid therapies. Inhibition of kinetoplast replication and expression is therefore an attractive target for the development of new therapies. In addition, the unique structure of the kinetoplast, which is composed of thousands of interlocked DNA rings, has attracted the interest of biophysicists and physical chemists as such ‘Olympic networks’ have unique material properties. However, the precise structure and biogenesis of the kinetoplast, and how these features relate to its function and physicochemical properties, have remained elusive.
By combining single-molecule imaging, sequencing, and mitochondrial DNA engineering, we are in a unique position to investigate kinetoplast biology in unprecedented detail. More specifically, by using mitochondrially targeted activator-like effector nucleases (mitoTALENS) developed in the Schnaufer lab, we will perform precisely controlled structural manipulations of mitochondrial DNA. In turn, we will use atomic force microscopy (AFM) and optical tweezers to characterise kDNA structure and topology at single-molecule detail and in response of the mitoTALENS manipulations. This single-molecule approach will be also complemented by kDNA deep sequencing and assembly tools, which will allow us to reveal the exact molecular composition of kDNA networks.
This project will build on these approaches to answer these questions:
- The biogenesis of kDNA is intimately linked to its replication and segregation mechanisms, which are largely mysterious. Using synchronised parasite cultures and specific probes for newly replicated molecules, the student will investigate the precise attachment sites and subsequent distribution of these molecules within the network during replication and dissect the process at single-molecule resolution.
- Anti-trypanosomatid drugs such as isometamidium, ethidium bromide and diamidine apparently kill parasites by inducing kDNA loss over time. However, the exact effects of these drugs on kDNA topology are unknown. The student will resolve this question by treating parasites with physiologically relevant drug concentrations, followed by single-molecule characterisation of kDNA topology (via AFM or optical tweezers).
Outcome: kDNA is essential for parasite survival and an established drug target. Yet, key aspects of kDNA topology / biogenesis and drug action remain unresolved. By bridging labs with leading expertise in kDNA biology and biophysics for the first time, this PhD project will start to close important gaps in our knowledge.
Training and skills development
The student will obtain cross-disciplinary training in cutting edge methods in molecular imaging (Atomic Force Microscopy and optical tweezers with a state-of-the-art LUMICK C-Trap), genetic engineering, and biophysics (Molecular Dynamics simulations and polymer physics modelling). This will provide them with a unique skill set which they could apply to various problems at the intersection of biology and physics, but in particular to the study of DNA and DNA-protein interactions in other organisms.
Beyond skills that are of obvious, direct and common benefit to both the individual researcher and the project, the student will be strongly encouraged to engage in training of ‘soft’ skills that will serve the researcher in the long run and help prepare them for future career opportunities. The University of Edinburgh, through its Institute for Academic Development (https://www.ed.ac.uk/institute-academic-development), provides ample opportunities for such training.
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