In the process of metastasis, cancer cells may transmigrate through the endothelium barrier of the vascular walls and into the circulatory blood system. During this process, the cancer cells...Show moreIn the process of metastasis, cancer cells may transmigrate through the endothelium barrier of the vascular walls and into the circulatory blood system. During this process, the cancer cells interact with the endothelial cells, resulting in the alternation of their mechanical properties. Although this interaction has been broadly studied from the perspective of cancer cells, no thorough investigation of the endothelial mechanical properties has been performed. In this thesis, by using a micro-rheology AFM-based approach, we show that the properties of endothelial cells change when cultured in cancer cell conditioned medium, as well as when in contact with cancer cells. We found that the stiffness of endothelial cells increased when cultured in a low-dilution cancer cell medium, while it decreased in high-dilution medium. This change was also evident on the viscoelasticity of the cells, with the endothelial cells cultured in high dilution medium showing lower viscoelastic properties. Furthermore, endothelial cells in direct contact with a cancer cell showed an increased height, as a result of the cancer cell's transmigration through the monolayer. Our findings demonstrate that the properties of endothelial cells change indirectly due to cancer cell secreted substances, and directly during the cancer cells' physical transmigration. This indicates that the endothelium is actively responding to the presence of cancer cells, rather than being a passive barrier as once believed.Show less
To better understand tumor progression and metastasis, it is important to investigate the mechanical properties of its cellular components. Tumors generally consist of cancer cells and healthy...Show moreTo better understand tumor progression and metastasis, it is important to investigate the mechanical properties of its cellular components. Tumors generally consist of cancer cells and healthy cells, whose interactions are fundamental for their structure and functionality. It has been shown that in co-cultured spheroids, cells rearrange themselves and completely separate. To closely mimic the tumor micro-environment in-vitro, hetero-spheroids containing both cancer cells and fibroblasts were used. The forces generated during cell-cell interaction and their cell sorting were studied. The interaction between the two cell types was probed with cell-sized (15-30 μm) microparticles. Different seeding-times and number ratios were investigated. No significant difference in the stress fields applied by the two different cell types during their interaction was found. However, it was observed that there is a critical number ratio between 1:3 and 1:6, above which the two cell types tend to completely separate. Below the critical ratio, there were intermixed areas of both cell lines. These cell clusters tend to merge over time, however no complete phase separation of the two cell types was observed for a period of one week. These results show that there is a favourable rearrangement of the cells consisting tumor-like structures. This cell type separation could indicate the next steps towards understanding the clustering and detachment of the cancer cells from the primary tumor, during metastasis.Show less
Current models of mitotic chromosome organization have focused mainly on the role that different the proteins have. However, recent studies have found that polyamines (PA) also have an effect on...Show moreCurrent models of mitotic chromosome organization have focused mainly on the role that different the proteins have. However, recent studies have found that polyamines (PA) also have an effect on the condensation of the mitotic chromosome. It was reported that without PA the mitotic chromosome swells and gains volume. When PA is reintroduced the initial volume is recovered. In this thesis, the effect of PA on the condensation of chromosomes was further investigated. First, the effect that changing the PA content of the buffer has on the entire mitotic chromosome was investigated. It was observed that when a chromosome is moved from a buffer with PA to a buffer without PA the length of the chromosome is increased. However, the reverse action was not observed. Next, the effect that PA has on protein rich regions of the mitotic chromosome was investigated. To this end, staining of a variety of proteins with immunofluorescence (IF) staining was tried. The proteins of interest were, centromere protein A (CENPA), NCAPH1, NCAPH2 and Topoisomerase IIa. Whilst early test of CENPA staining were successful this result was not reproducible when new antibodies were ordered. Furthermore, the staining of the other proteins was also unsuccessful. All of the unsuccessful IF staining showed a lack of specificity since most of the chromosomes were stained instead of the specific regions were the proteins are known to aggregate. Since no successful staining of proteins could be performed, it was not possible to measure the effects that polyamines have on the regions were CENPA, NCAPH1, NCAPH2 and Topoisomerase IIa bind.Show less
Fluorescence Correlation Spectroscopy (FCS) and F¨orster Resonance Energy Transfer (FRET) are used in combination to study conformational dynamics of biological complexes such as nucleosomes....Show moreFluorescence Correlation Spectroscopy (FCS) and F¨orster Resonance Energy Transfer (FRET) are used in combination to study conformational dynamics of biological complexes such as nucleosomes. However, it is currently unclear how experimental conditions affect the accuracy with which rates of conformational dynamics are determined. We develop a computational method that allows us to simulate FCS data for FRET labelled molecules under Pulsed Interleaved Excitation (PIE). Using these simulations, we determined the effect of experimental conditions such as laser intensity, measurement time, sample concentration and shift in laser focus on the correlation curves and extracted diffusion time and rate of transitions. We show two ways to accurately determine transition rates of diffusing molecules in single experiments. One using PIE and a wavelength dependent correction factor and another under continuous excitation. These simulations can assist in obtaining new insight on protein- DNA interaction studies from combined PIE FRET-FCS studies.Show less
The hydrodynamic properties of self-propelled particles have, in the past, been approximated by a multipole expansion. As it becomes easier to produce synthetic swimmers in various shapes and sizes...Show moreThe hydrodynamic properties of self-propelled particles have, in the past, been approximated by a multipole expansion. As it becomes easier to produce synthetic swimmers in various shapes and sizes, a proper understanding of this hydrodynamic basis will likely be important. In this thesis, simulation was used to explore the diusive behavior of the first-order term in the presence of a wall. To quantify this behavior, the characteristic rotation time and eective diusion coecient were determined for a range of values of the first-order expansion coecient . It is shown that for low the particle diuses in three dimensions, while for high alpha it diuses in two dimensions as it is locked parallel to the wall.Show less
Doxorubicine and Aclarubicine are widely used anti-tumor medicine that intercalate in betweenDNA basepairs. Exactly how these medicines get their anti-tumor behaviour is still a subject fordebate....Show moreDoxorubicine and Aclarubicine are widely used anti-tumor medicine that intercalate in betweenDNA basepairs. Exactly how these medicines get their anti-tumor behaviour is still a subject fordebate. In an effort to understand the anti-tumor behaviour of these medicines better, the effectsof intercalation on the intrinsic twist and the twist modulus of DNA was investigated. Changesto these two factors have effects on the way the DNA compacted through supercoiling whichcould have effects on the accessibility of genes. It was found that both medicines cause a similarunwinding of the double helix namely, 10.7°±0.8°for Doxorubicine and 14.1°±0.6°for Aclaru-bicine. Furthermore, the twist modulus was decreased significantly from 100 nm in naked DNAto 40 nm±2 nm with Doxorubicine bound and 44 nm±3 nm with Aclarubicine bound. Ad-ditionally, bound intercalators increase the force necessary to melt DNA, which could inhibitcellular processes such as DNA replication. Therefore, to investigate the binding strength ofDoxorubicine and Aclarubicine a force spectroscopy experiment was prepared with a hairpinon a custom made flow cell. While this hairpin construction is still a work in progress, workon creating a hairpin out of sequenced DNA on a Miseq flow cell was started. A hairpin de-sign was tested that altered the linear sequenced DNA into a hairpin construct by restrictingthe DNA and ligating a small hairpin oligo. An azide-nucleotide was built in and throughstrain-promoted azide-alkyne cycloaddition a long tether with a paramagnetic bead was at-tached. With such new hairpins generated from sequenced DNA, the sequence specific bindingstrength of intercalators could be investigated. On top of this, the hairpins could be used toinvestigate the sequence specificity of a wide range of DNA binders.Show less
The cytoskeleton gives a cell its main structure and rigidity. It plays a significant role during many (force sensitive) mechanical cues from outside the cell. The anisotropy of the cytoskeleton...Show moreThe cytoskeleton gives a cell its main structure and rigidity. It plays a significant role during many (force sensitive) mechanical cues from outside the cell. The anisotropy of the cytoskeleton has been shown to control the geometry and forces of adherent cells. Bases on the the shape of curved cell edge segments of single cells, the size of internal stresses and traction forces can be calculated. However, such methods have not yet been applied to clusters of cells. Here we introduce two methods to describe the mechanical equilibrium of cell doublets. Either in a discontinuous way, where both cells are treated separately, or continuous, where we do not differentiate between the cells and treat it as a single cell.Show less
In this thesis we investigate the effects of bending on a Miura Ori Origami sheet. In particular we are going to analyse a simplified version, a single stripe made by a row of unit cells. We will...Show moreIn this thesis we investigate the effects of bending on a Miura Ori Origami sheet. In particular we are going to analyse a simplified version, a single stripe made by a row of unit cells. We will investigate how the bending stiffness and the curvature at the surface are changing with the variation of the fold angle of the structure and with the application of an external force. We investigate the curvature by scanning the samples with a 3D scanner and then analysing the data. To probe the bending stiffness, we use an Instron, a uniaxial compression device.Show less
The nucleosome consists of a short stretch of DNA wrapped around a protein cylinder, and is the fundamental unit of chromatin, which compacts the DNA into the cell nucleus. The nucleosome is known...Show moreThe nucleosome consists of a short stretch of DNA wrapped around a protein cylinder, and is the fundamental unit of chromatin, which compacts the DNA into the cell nucleus. The nucleosome is known to transiently partially unwrap or 'breathe' \textit{in vitro}, exposing DNA which would otherwise be sterically inaccessible to enzymes. Breathing is investigated for its potential importance \textit{in vivo} in both essential DNA processes, and in higher-order chromatin organisation. In this thesis we present a two-parameter physical statistical model of the breathing process based on steric enzyme accessibility, the energetics of the bent DNA molecule, and the adsorption of the DNA upon the proteins. We estimate the elastic energy using Monte Carlo simulations of a coarse-grained model of the nucleosomal DNA, and we fit the model to the available experimental results. We find in agreement with experimental studies that site accessibility decays exponentially toward the centre sites, and that highly asymmetric breathing behaviour is possible due to the very sensitive dependence of breathing upon energy distribution, and in turn, sequence.Show less
Metamaterials exhibit exotic properties derived from their geometric structures. A procedure has recently been developed to build reconfigurable structures from convex polyhedra. While initally...Show moreMetamaterials exhibit exotic properties derived from their geometric structures. A procedure has recently been developed to build reconfigurable structures from convex polyhedra. While initally characterized as rigid, we found that some of these structures possess multiple stable configurations. In the present study we develop numerical tools to simulate all possible deformations that can be applied to these structures, mapping the corresponding energy landscape. We use the simulation to identify the additional stable configurations and study their dependence on key physical parameters. Based on the results found, we explain the mechanism behind the observed multistability and suggest its validity as fundamental ingredient for a general designing rule. Finally, we explore the possibility of implementing our results into the development of a reconfigurable, multistable and multifunctional 3D material.Show less
In recent years, a number of artificial materials (so called metamaterials) have been developed, tailoring exotic responses to mechanical stress by exploiting geometrical features. A particular...Show moreIn recent years, a number of artificial materials (so called metamaterials) have been developed, tailoring exotic responses to mechanical stress by exploiting geometrical features. A particular class of 2D metamaterials consists of tilings of polygons that can fold onto themselves by exploiting a mechanical instability triggered by compression. A subclass of these materials shows a sequence of two ordered states (one partially and one fully closed) when biaxially compressed; however, the reason why such networks fold in multiple steps is yet elusive and we are unaware of a general rule to design them; it is even unknown whether it is possible to increase the number of ordered states sequentially reached by a network during the folding process. In order to answer these questions, first we investigate a variety of designs to determine the role played by symmetries, topology and elastic energy; secondly, we focus on a hierarchical approach to design a network with three folding stages; finally, we fully characterize the mechanical response of this new network and we study how the order of the system evolves during the folding process.Show less
Complex cellular processes can be characterised using single particle tracking techniques (SPT). ‘Labels’ such as metal nanoparticles are introduced into cells and tracked to reveal molecular...Show moreComplex cellular processes can be characterised using single particle tracking techniques (SPT). ‘Labels’ such as metal nanoparticles are introduced into cells and tracked to reveal molecular trajectories. Many current techniques are based on fluorescence microscopy, and have nanometre-resolution. To accurately probe cellular processes, a technique must also have long-term 3D in-vivo observing capability with a minimal toxic effect. Gold nanorods (GNRs) in two-photon microscopy is a promising technique. GNRs are non-toxic, easily functionalisable, and exhibit a bright two-photon fluorescence. However, the theoretical positional accuracy for this technique is not yet known. Furthermore, the detailed trajectory data present statistical challenges. We have addressed these issues here numerically, using simulated image data. We found the accuracy to be between 5.2nm and 8.9nm for stationary GNRS, dependent on the separation between slices. We also found that diffusive movement of a nanorod lowers the accuracy, at worst case to 31.8nm. We have also investigated optimising the extraction of behaviour properties from MSD plots, and have used Welsh’s test to detect transitions. GNRs in two-photon microscopy has been shown to be a very accurate technique, and its trajectory data can yield accurate behavioural information. The fit of the PSF may be improved, but the techniques already compares well against others.Show less
