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Research Experience

MOAC MSc Mini-projects:

"Purification of Non-Neuronal Human Tubulin from Human Embryonic Kidney Cells"

Supervisor: Anne Straube

As part of my Experimental Biology Project, I assisted in the development of a method for purification of non-neuronal tubulin from human embryonic kidney (HEK) cells. Conventional protocols use differential centrifugation to purify tubulin polymers (microtubules) from neuronal tissue, such as pig brain. These techniques exploit the abundance of fast polymerizing β2 and β3 tubulin isotypes within the neuronal tissue to achieve large yields. However, non-neuronal cell types express comparatively small amounts of tubulin, most of which consists of slow - polymerising β1 and β4 isotypes. Thus, I analysed the efficaciousness of a range of established methods for the purification of tubulin from HEK cells. I also learnt to use TIRF for observing in-vitro polymerisation of tubulin.

"Linear Dichroism: A Tool for Determining The Orientation of Lipid Probes within Liposomes and Lipid Discs"

Supervisors: Nikola Chmel and Alison Rodger

In my Experimental Biophysical Project, I designed and developed a set of tools that could be used to determine the 3D orientation of lipid probes, within liposomes and lipid discs, via the use of linear Dichroism (LD). We were able to use the toolset to determine the 3D orientation of a range of lipid probes across a range of substrate supports of varying hydrophobicity, to a first approximation.

"Investigating Relationships between Tissue Flow, Cell Size, and Embryo Topology during Chick Gastrulation"

Supervisor: Till Bretschneider

In my Theoretical Project, I was given the opportunity to develop scripts in MATLAB for data analysis. A watershed segmentation algorithm, developed by the Bretschneider group, was used to segment cells within the ectoderm of a chick embryo, during gastrulation. I thence developed MATLAB scripts that were used to calculate the 3D velocity and curvature of trajectories for segmented cells, as well as measure changes in cell size and shape.


Other Research Projects:

URSS project

Supervisor: Corinne Smith

In this project, I investigated the effectiveness of graphene as a tool to obtain higher contrast images of 70 kDa and 190 kDa proteins by transmission electron microscopy (TEM), without the use of heavy metal salts such as uranyl acetate. Graphene and graphene oxide (GO) were used as specimen supports, and an alternative to amorphous carbon, which is the traditional specimen support used in TEM. We aimed to exploit the relative thinness of graphene to reduce elastic and inelastic electron scattering of TEM beam electrons by the support in order to obtain higher contrast images. Unfortunately, graphene supports offered little improvement in contrast. I also learnt to purify clathrin from pig brain.

Dissertation

Supervisor: Vilmos Fülöp

As part of my dissertation, I modeled the three-dimensional structure of Trypanosoma brucei Oligopeptidase B (OPB) in its open conformational state. Multi-wavelength anomalous dispersion of selenomethionine-substituted protein crystals were used to solve the phase problem. I used non-crystallographic symmetry averaging to refine the phase information and the electron density map. Through structural refinement I achieved a significant improvement in the crystallographic residual factor with little change in the free residual factor, indicating a good fit between the model and experimental data. I also evaluated the structural analogy of T. brucei OPB with other members of the prolyl oligopeptidase family through structural alignment, as well as a comparison of structural motifs and active sites.

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Contact Details

MOAC Doctoral Training Centre
University of Warwick
Coventry, CV4 7AL, UK

S dot T dot Clarke at warwick dot ac dot uk


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