The influence of pH and fluid dynamics on the antibacterial efficacy of 45S5 Bioglass

In recent years, there has been considerable interest in the potential antibacterial properties that bioactive glasses may possess. However, there have been several conflicting reports on the antibacterial efficacy of 45S5 Bioglass®. Various mechanisms regarding its mode of action have been proposed, such as changes in the environmental pH, increased osmotic pressure, and ‘needle like’ sharp glass debris which could potentially damage prokaryotic cell walls and thus inactivate bacteria. In this current study, a systematic investigation was undertaken on the antibacterial efficacy of 45S5 Bioglass® on Escherichia coli NCTC 10538 and Staphylococcus aureus ATCO 6538 under a range of clinically relevant scenarios including varying Bioglass® concentration, direct and indirect contact between Bioglass® and microorganisms, static and shaking incubation conditions, elevated and neutralised pH environments. The results demonstrated that under elevated pH conditions, Bioglass® particles has no antibacterial effect on S. aureus whilst, a concentration dependent antibacterial effect against E. coli was observed. However, the antibacterial activity ceased when the pH of the media was neutralised. The results of this current study therefore suggest that the mechanism of antibacterial activity of Bioglass® is associated with changes in the environmental pH; an environment that is less likely to occur in vivo due to buffering of the system.

Use of imaging technology to better understand soft contact lens fit dynamics

The principal theme of this thesis is the identification of additional factors affecting, and consequently to better allow, the prediction of soft contact lens fit. Various models have been put forward in an attempt to predict the parameters that influence soft contact lens fit dynamics; however, the factors that influence variation in soft lens fit are still not fully understood. The investigations in this body of work involved the use of a variety of different imaging techniques to both quantify the anterior ocular topography and assess lens fit. The use of Anterior-Segment Optical Coherence Tomography (AS-OCT) allowed for a more complete characterisation of the cornea and corneoscleral profile (CSP) than either conventional keratometry or videokeratoscopy alone, and for the collection of normative data relating to the CSP for a substantial sample size. The scleral face was identified as being rotationally asymmetric, the mean corneoscleral junction (CSJ) angle being sharpest nasally and becoming progressively flatter at the temporal, inferior and superior limbal junctions. Additionally, 77% of all CSJ angles were within ±50 of 1800, demonstrating an almost tangential extension of the cornea to form the paralimbal sclera. Use of AS-OCT allowed for a more robust determination of corneal diameter than that of white-to-white (WTW) measurement, which is highly variable and dependent on changes in peripheral corneal transparency. Significant differences in ocular topography were found between different ethnicities and sexes, most notably for corneal diameter and corneal sagittal height variables. Lens tightness was found to be significantly correlated with the difference between horizontal CSJ angles (r =+0.40, P =0.0086). Modelling of the CSP data gained allowed for prediction of up to 24% of the variance in contact lens fit; however, it was likely that stronger associations and an increase in the modelled prediction of variance in fit may have occurred had an objective method of lens fit assessment have been made. A subsequent investigation to determine the validity and repeatability of objective contact lens fit assessment using digital video capture showed no significant benefit over subjective evaluation. The technique, however, was employed in the ensuing investigation to show significant changes in lens fit between 8 hours (the longest duration of wear previously examined) and 16 hours, demonstrating that wearing time is an additional factor driving lens fit dynamics. The modelling of data from enhanced videokeratoscopy composite maps alone allowed for up to 77% of the variance in soft contact lens fit, and up to almost 90% to be predicted when used in conjunction with OCT. The investigations provided further insight into the ocular topography and factors affecting soft contact lens fit.

The structure and dynamics of saxicolous lichen communities

The saxicolous lichen vegetation on Ordovician slate rock at the mouth of the River Dovey, South Merionethshire, Wales was described in relation to several environmental variables which include aspect, slope angle, light intensity, rock porosity, rock microtopography and rock stability. Each of the measured environmental variables was shown to influence the lichen vegetation. A number of groups of species which were characteristic of certain environments were described. The data from the saxicolous lichen communities were analysed using multivariate analysis. Qualitative and quantitative data were ordinated, the qualitative data being easier to interpret ecologically, and site number (which reflects distance from the sea and altitude), rock porosity and light intensity were shown to be important environmental variables. A classification of the data was also carried out. The results of the ordination and classification were combined together and a model constructed which describes saxicolous lichen vegetation. A method which uses the model as an aid to the design and interpretation of field experiments is described. The model is applied to an experiment which investigates the effect on growth of transplanting four saxicolous lichens to different aspects. Growth was inhibited in Physcia orbicularis and Parmelia conspersa on rock surfaces of northwest aspect compared with growth on rock surfaces of southeast aspect. Growth was inhibited in Parmelia glabratula ssp. fuliginosa on rock surfaces of southeast aspect compared with rock surfaces of northwesr aspect. The growth of Parmelia saxatilis was similar at both southeast and northwesr aspects. Growth inhibition or stimulation in thalli of Physcia orbicularis, Parmelia conspersa and Parmelia glabratula ssp. fuliginosa after transplantation was consistent with the predictions of the model while the results for Parmelia saxatilis were not as expected. There was evidence that the frequency of Parmelia conspersa and Parmelia glabratula at a site is related to an effect of the environment on the growth of the thalli. There was also evidence that the frequency of Physcia orbicularis at a site is related to an effect of the environment on the establishment phase of the thalli and for the competitive exclusion of Parmelia saxatilis thalli from southeast facing rock surfaces. The distribution of lichens in relation to height on nine rock surfaces was investigated. It was suggested that the distribution of the lichens was influenced by microclimatic factors which are related to height on the rock, environmental variables which are associated with the rock substratum (e.g. rock porosity and rock microtopography) and by historical factors. The pattern of one crustose and one foliose lichen on four rock surfaces of different aspect and slope was investigated. On the vertically inclined surface the density of small thalli of Buellia aethalea and Parmelia glabratula ssp fuliginosa was correlated with the microtopography of the surface in transects horizontally across the rock surface but not in transects vertically down the rock surface. there were consitent differences in the scale and intensity of pattern horizontally and vertically and also a decrease in the intensity of pattern vertically as the slope of the rock surface decreased. These results were consistent with the suggestion of a gradient of microclimatic factors up the rock. The differences in the scale and intensity of pattern in different size classes in the population were consistent with the changes in pattern with time which have been shown to occur during succession in sand dune and salt marsh vegetation. The relationship between thallus size and height on a rock surface and between the radial growth rate and location of a thallus on a rock surface were investigated. Thalli of Parmelia glabratula ssp. fuliginosa were larger at the top of the rock surface than at the bottom and the data were consistent with the suggestion that the colonisation of the rock surface began at the top and, in time, spread downwards. The radial growth rate of the thalli could not be related to variation in slope, porosity, microtopography or directly to height on the rock but could be related to the horizontal location of the thalli on the rock. These results were consistent with the suggestion that here is a gradient of microclimatic factors across the rock surface which is also modified by height on the rock surface. The succession of lichen communities was described by relating the vegetation to rock porosity, rock microtopography, species diversity and rock stability. An initial stage dominated by crustose lichens leads to communities dominated by crustose, foliose and fruticose species. In the late stages of the succession on some rock surfaces crustose species again become dominant. The occurrence of the climax state and cyclic vegetation change in lichen communities are discussed. A mthod of estimating the age structure of a lichen population by relating thallus size to growth rate is described. The sources of error in the method are discussed in some detail and several refinements suggested to increase the accuracy of the method. The population dynamics of Parmelia glabratula ssp. fuliginosa was investigated by applying life tables to the age structures of eight different populations. The data were consistent with a period of relatively constant recruitment of thalli into the populations. Mortality in lichen populations was divided into deaths which occur after fragmentation of the thallus and deaths which occur after catastrophic environmental events. THe data suggest that the rate of fragmenting death is dependent on the age of the thallus while the rate of catastrophic death is dependent on the number of thalli established in an age class. A comparison of the numbers of thalli in each age class in the eight populations suggested that population density is controlled firstly, by climate and secondly, by variables related to the local rock surface environment. The rate of fragmenting death is related to the diversity of the community and the influence of diversity together with environmental variables in fluctuating or cyclic changes in population number.

Network evolution and the spatiotemporal dynamics of knowledge sourcing

Knowledge accessing from external organisations is important to firms, especially entrepreneurial ones which often cannot generate internally all the knowledge necessary for innovation. There is, however, a lack of evidence concerning the association between the evolution of firms and the evolution of their networks. The aim of this paper is to begin to fill this gap by undertaking an exploratory analysis of the relationship between the vintage of firms and their knowledge sourcing networks. Drawing on an analysis of firms in the UK, the paper finds some evidence of a U-shaped relationship existing between firm age and the frequency of accessing knowledge from certain sources. Emerging entrepreneurial firms tend to be highly active with regard to accessing knowledge for a range of sources and geographic locations, with the rate of networking dropping somewhat during the period of peak firm growth. For instance, it is found that firms tend to less frequently access knowledge sources such as universities and research institutes in their own region during a stage of peak turnover growth. Overall, the results suggest a complex relationship between the lifecycle of the firm and its networking patterns. It is concluded that policymakers need to become more aware that network formation and utilisation by firms is likely to vary dependent upon their lifecycle position.

Thermalized polarization dynamics of a discrete optical-waveguide system with four-wave mixing

Statistical mechanics of two coupled vector fields is studied in the tight-binding model that describes propagation of polarized light in discrete waveguides in the presence of the four-wave mixing. The energy and power conservation laws enable the formulation of the equilibrium properties of the polarization state in terms of the Gibbs measure with positive temperature. The transition line T=∞ is established beyond which the discrete vector solitons are created. Also in the limit of the large nonlinearity an analytical expression for the distribution of Stokes parameters is obtained, which is found to be dependent only on the statistical properties of the initial polarization state and not on the strength of nonlinearity. The evolution of the system to the final equilibrium state is shown to pass through the intermediate stage when the energy exchange between the waveguides is still negligible. The distribution of the Stokes parameters in this regime has a complex multimodal structure strongly dependent on the nonlinear coupling coefficients and the initial conditions.

Seasonal dynamics of an aquatic macroinvertebrate assembly (Hydrobiological case study of Lake Balaton No. 2)

In 2002, 2003 and 2004, we took macoinvertebrate samples on a total of 36 occasions at the Badacsony bay of Lake Balaton. Our sampling site was characterised by areas of open water (in 2003 and 2004 full of reed-grass) as well as by areas covered by common reed (Phragmites australis) and narrowleaf cattail (Typha angustifolia). Samples were taken both from water body and benthic ooze by use of a stiff hand net. We have gained our data from processing 208 individual samples. We took samples frequently from early spring until late autumn for a deeper understanding of the processes of seasonal dynamics. The main seasonal patterns and temporal changes of diversity were described. We constructed a weather-dependent simulation model of the processes of seasonal dynamics in the interest of a possible further utilization of our data in climate change research. We described the total number of individuals, biovolume and diversity of all macroinvertebrate species with a single index and used the temporal trends of this index for simulation modelling. Our discrete deterministic model includes only the impact of temperature, other interactions might only appear concealed. Running the model for different climate change scenarios it became possible to estimate conditions for the 2070-2100 period. The results, however, should be treated very prudently not only because our model is very simple but also because the scenarios are the results of different models.

A nanoliter volume nuclear magnetic resonance (NMR) system using tunneling magneto-resistive (TMR) sensors to recognize biomolecules

The need to incorporate advanced engineering tools in biology, biochemistry and medicine is in great demand. Many of the existing instruments and tools are usually expensive and require special facilities.^ With the advent of nanotechnology in the past decade, new approaches to develop devices and tools have been generated by academia and industry. ^ One such technology, NMR spectroscopy, has been used by biochemists for more than 2 decades to study the molecular structure of chemical compounds. However, NMR spectrometers are very expensive and require special laboratory rooms for their proper operation. High magnetic fields with strengths in the order of several Tesla make these instruments unaffordable to most research groups.^ This doctoral research proposes a new technology to develop NMR spectrometers that can operate at field strengths of less than 0.5 Tesla using an inexpensive permanent magnet and spin dependent nanoscale magnetic devices. This portable NMR system is intended to analyze samples as small as a few nanoliters.^ The main problem to resolve when downscaling the variables is to obtain an NMR signal with high Signal-To-Noise-Ratio (SNR). A special Tunneling Magneto-Resistive (TMR) sensor design was developed to achieve this goal. The minimum specifications for each component of the proposed NMR system were established. A complete NMR system was designed based on these minimum requirements. The goat was always to find cost effective realistic components. The novel design of the NMR system uses technologies such as Direct Digital Synthesis (DDS), Digital Signal Processing (DSP) and a special Backpropagation Neural Network that finds the best match of the NMR spectrum. The system was designed, calculated and simulated with excellent results.^ In addition, a general method to design TMR Sensors was developed. The technique was automated and a computer program was written to help the designer perform this task interactively.^

The potential role of plant oxygen and sulphide dynamics in die-off events of the tropical seagrass, Thalassia testudinum

1 Oxygen and sulphide dynamics were examined, using microelectrode techniques, in meristems and rhizomes of the seagrass Thalassia testudinum at three different sites in Florida Bay, and in the laboratory, to evaluate the potential role of internal oxygen variability and sulphide invasion in episodes of sudden die-off. The sites differed with respect to shoot density and sediment composition, with an active die-off occurring at only one of the sites. 2 Meristematic oxygen content followed similar diel patterns at all sites with high oxygen content during the day and hyposaturation relative to the water column during the night. Minimum meristematic oxygen content was recorded around sunrise and varied among sites, with values close to zero at the die-off site. 3 Gaseous sulphide was detected within the sediment at all sites but at different concentrations among sites and within the die-off site. Spontaneous invasion of sulphide into Thalassia rhizomes was recorded at low internal oxygen partial pressure during darkness at the die-off site. 4 A laboratory experiment showed that the internal oxygen dynamics depended on light availability, and hence plant photosynthesis, and on the oxygen content of the water column controlling passive oxygen diffusion from water column to leaves and belowground tissues in the dark. 5 Sulphide invasion only occurred at low internal oxygen content, and the rate of invasion was highly dependent on the oxygen supply to roots and rhizomes. Sulphide was slowly depleted from the tissues when high oxygen partial pressures were re-established through leaf photosynthesis. Coexistence of sulphide and oxygen in the tissues and the slow rate of sulphide depletion suggest that sulphide reoxidation is not biologically mediated within the tissues of Thalassia. 6 Our results support the hypothesis that internal oxygen stress, caused by low water column oxygen content or poor plant performance governed by other environmental factors, allows invasion of sulphide and that the internal plant oxygen and sulphide dynamics potentially are key factors in the episodes of sudden die-off in beds of Thalassia testudinum . Root anoxia followed by sulphide invasion may be a more general mechanism determining the growth and survival of other rooted plants in sulphate-rich aquatic environments.

History and evolutionary trajectory of the Iranian nuclear program

What actors and processes at what levels of analysis and through what mechanisms have pushed Iran's nuclear program (INP) towards being designated as a proliferation threat (securitization)? What actors and processes at what levels of analysis and through what mechanisms have pushed Iran's nuclear program away from being designated as an existential threat (de-securitization)? What has been the overall balance of power and interaction dynamics of these opposing forces over the last half-century and what is their most likely future trajectory? ^ Iran's nuclear story can be told as the unfolding of constant interaction between state and non-state forces of "nuclear securitization" and "nuclear de-securitization." Tracking the crisscrossing interaction between these different securitizing and de-securitizing actors in a historical context constitutes the central task of this project. ^ A careful tracing of "security events" on different analytical levels reveals the broad contours of the evolutionary trajectory of INP and its possible future path(s). Out of this theoretically conscious historical narrative, one can make informed observations about the overall thrust of INP along the securitization - de-securitization continuum. ^ The main contributions of this work are three fold: First, it brings a fresh theoretical perspective on Iran's proliferation behavior by utilizing the "securitization" theory tracing the initial indications of the threat designation of INP all the way back to the mid 1970s. Second, it gives a solid and thematically grounded historical texture to INP by providing an intimate engagement with the persons, processes, and events of Tehran's nuclear pursuit over half a century. Third, it demonstrates how INP has interacted with and even at times transformed the NPT as the keystone of the non-proliferation regime, and how it has affected and injected urgency to the international discourse on nuclear proliferation specifically in the Middle East.^

Effects of seasonally varying temperature and salinity on the dynamics of sea lice (Lepeophtheirus salmonis)

Sea lice (Lepeophtheirus salmonis) are an economically significant parasite in salmonid aquaculture. They exhibit temperature-dependent development rates and salinity-dependent mortality, which can greatly impact sea lice population dynamics, but no deterministic models have incorporated these seasonal variables. To understand how seasonality affects sea lice population dynamics, I derive a delay differential equation model with temperature and salinity dependence. I find that peak reproductive output in Newfoundland and British Columbia differs by four months. A sensitivity analysis shows sea lice abundance is most sensitive to variation in mean annual water temperature and salinity, whereas it is lease sensitive to infection rate. Additionally, I investigate the effects of production cycle timing on sea lice management and find that optimal production cycle start times are between the 281st and 337th days of the year in Newfoundland. I also demonstrate that adjusting follow-up treatment timing in response to temperature can improve treatment regimes. My results suggest that effective sea lice management requires consideration of local temperature and salinity patterns.

Dynamics of spatial heterogeneity in a landfill with interacting phase densities:a stochastic analysis

A landfill represents a complex and dynamically evolving structure that can be stochastically perturbed by exogenous factors. Both thermodynamic (equilibrium) and time varying (non-steady state) properties of a landfill are affected by spatially heterogenous and nonlinear subprocesses that combine with constraining initial and boundary conditions arising from the associated surroundings. While multiple approaches have been made to model landfill statistics by incorporating spatially dependent parameters on the one hand (data based approach) and continuum dynamical mass-balance equations on the other (equation based modelling), practically no attempt has been made to amalgamate these two approaches while also incorporating inherent stochastically induced fluctuations affecting the process overall. In this article, we will implement a minimalist scheme of modelling the time evolution of a realistic three dimensional landfill through a reaction-diffusion based approach, focusing on the coupled interactions of four key variables - solid mass density, hydrolysed mass density, acetogenic mass density and methanogenic mass density, that themselves are stochastically affected by fluctuations, coupled with diffusive relaxation of the individual densities, in ambient surroundings. Our results indicate that close to the linearly stable limit, the large time steady state properties, arising out of a series of complex coupled interactions between the stochastically driven variables, are scarcely affected by the biochemical growth-decay statistics. Our results clearly show that an equilibrium landfill structure is primarily determined by the solid and hydrolysed mass densities only rendering the other variables as statistically "irrelevant" in this (large time) asymptotic limit. The other major implication of incorporation of stochasticity in the landfill evolution dynamics is in the hugely reduced production times of the plants that are now approximately 20-30 years instead of the previous deterministic model predictions of 50 years and above. The predictions from this stochastic model are in conformity with available experimental observations.

Determination of Biomolecular Interdomain Motions using Nuclear Magnetic Resonance

Biological macromolecules can rearrange interdomain orientations when binding to various partners. Interdomain dynamics serve as a molecular mechanism to guide the transitions between orientations. However, our understanding of interdomain dynamics is limited because a useful description of interdomain motions requires an estimate of the probabilities of interdomain conformations, increasing complexity of the problem.

Staphylococcal protein A (SpA) has five tandem protein-binding domains and four interdomain linkers. The domains enable Staphylococcus aureus to evade the host immune system by binding to multiple host proteins including antibodies. Here, I present a study of the interdomain motions of two adjacent domains in SpA. NMR spin relaxation experiments identified a 6-residue flexible interdomain linker and interdomain motions. To quantify the anisotropy of the distribution of interdomain orientations, we measured residual dipolar couplings (RDCs) from the two domains with multiple alignments. The N-terminal domain was directly aligned by a lanthanide ion and not influenced by interdomain motions, so it acted as a reference frame to achieve motional decoupling. We also applied {\it de novo} methods to extract spatial dynamic information from RDCs and represent interdomain motions as a continuous distribution on the 3D rotational space. Significant anisotropy was observed in the distribution, indicating the motion populates some interdomain orientations more than others. Statistical thermodynamic analysis of the observed orientational distribution suggests that it is among the energetically most favorable orientational distributions for binding to antibodies. Thus, the affinity is enhanced by a pre-posed distribution of interdomain orientations while maintaining the flexibility required for function.

The protocol described above can be applied to other biological systems in general. Protein molecule calmodulin and RNA molecule trans-activation response element (TAR) also have intensive interdomain motions with relative small intradomain dynamics. Their interdomain motions were studied using our method based on published RDC data. Our results were consistent with literature results in general. The differences could be due to previous studies' use of physical models, which contain assumptions about potential energy and thus introduced non-experimental information into the interpretations.

Drivers of Dengue Within-Host Dynamics and Virulence Evolution

Dengue is an important vector-borne virus that infects on the order of 400 million individuals per year. Infection with one of the virus's four serotypes (denoted DENV-1 to 4) may be silent, result in symptomatic dengue 'breakbone' fever, or develop into the more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Extensive research has therefore focused on identifying factors that influence dengue infection outcomes. It has been well-documented through epidemiological studies that DHF is most likely to result from a secondary heterologous infection, and that individuals experiencing a DENV-2 or DENV-3 infection typically are more likely to present with more severe dengue disease than those individuals experiencing a DENV-1 or DENV-4 infection. However, a mechanistic understanding of how these risk factors affect disease outcomes, and further, how the virus's ability to evolve these mechanisms will affect disease severity patterns over time, is lacking. In the second chapter of my dissertation, I formulate mechanistic mathematical models of primary and secondary dengue infections that describe how the dengue virus interacts with the immune response and the results of this interaction on the risk of developing severe dengue disease. I show that only the innate immune response is needed to reproduce characteristic features of a primary infection whereas the adaptive immune response is needed to reproduce characteristic features of a secondary dengue infection. I then add to these models a quantitative measure of disease severity that assumes immunopathology, and analyze the effectiveness of virological indicators of disease severity. In the third chapter of my dissertation, I then statistically fit these mathematical models to viral load data of dengue patients to understand the mechanisms that drive variation in viral load. I specifically consider the roles that immune status, clinical disease manifestation, and serotype may play in explaining viral load variation observed across the patients. With this analysis, I show that there is statistical support for the theory of antibody dependent enhancement in the development of severe disease in secondary dengue infections and that there is statistical support for serotype-specific differences in viral infectivity rates, with infectivity rates of DENV-2 and DENV-3 exceeding those of DENV-1. In the fourth chapter of my dissertation, I integrate these within-host models with a vector-borne epidemiological model to understand the potential for virulence evolution in dengue. Critically, I show that dengue is expected to evolve towards intermediate virulence, and that the optimal virulence of the virus depends strongly on the number of serotypes that co-circulate. Together, these dissertation chapters show that dengue viral load dynamics provide insight into the within-host mechanisms driving differences in dengue disease patterns and that these mechanisms have important implications for dengue virulence evolution.

Spatiotemporal Dynamics of CaMKI During Structural Plasticity of Single Dendritic Spines

Multifunctional calcium/calmodulin dependent protein kinases (CaMKs) are key regulators of spine structural plasticity and long-term potentiation (LTP) in neurons. CaMKs have promiscuous and overlapping substrate recognition motifs, and are distinguished in their regulatory role based on differences in the spatiotemporal dynamics of activity. While the function and activity of CaMKII in synaptic plasticity has been extensively studied, that of CaMKI, another major class of CaMK required for LTP, still remain elusive.

Here, we develop a Förster’s Resonance Energy Transfer (FRET) based sensor to measure the spatiotemporal activity dynamics of CaMK1. We monitored CaMKI activity using 2-photon fluorescence lifetime imaging, while inducing LTP in single dendritic spines of rat (Rattus Norvegicus, strain Sprague Dawley) hippocampal CA1 pyramidal neurons using 2-photon glutamate uncaging. Using RNA-interference and pharmacological means, we also characterize the role of CaMKI during spine structural plasticity.

We found that CaMKI was rapidly and transiently activated with a rise time of ~0.3 s and decay time of ~1 s in response to each uncaging pulse. Activity of CaMKI spread out of the spine. Phosphorylation of CaMKI by CaMKK was required for this spreading and for the initial phase of structural LTP. Combined with previous data showing that CaMKII is restricted to the stimulated spine and required for long-term maintenance of structural LTP, these results suggest that CaMK diversity allows the same incoming signal – calcium – to independently regulate distinct phases of LTP by activating different CaMKs with distinct spatiotemporal dynamics.

Role of Vinculin in Regulating Force-Sensitive Dynamics of Adherens Junctions During Collective Cell Migration

Dynamic processes such as morphogenesis and tissue patterning require the precise control of many cellular processes, especially cell migration. Historically, these processes are thought to be mediated by genetic and biochemical signaling pathways. However, recent advances have unraveled a previously unappreciated role of mechanical forces in regulating these homeostatic processes in of multicellular systems. In multicellular systems cells adhere to both deformable extracellular matrix (ECM) and other cells, which are sources of applied forces and means of mechanical support. Cells detect and respond to these mechanical signals through a poorly understood process called mechanotransduction, which can have profound effects on processes such as cell migration. These effects are largely mediated by the sub cellular structures that link cells to the ECM, called focal adhesions (FAs), or cells to other cells, termed adherens junctions (AJs).

Overall this thesis is comprised of my work on identifying a novel force dependent function of vinculin, a protein which resides in both FAs and AJs - in dynamic process of collective migration. Using a collective migration assay as a model for collective cell behavior and a fluorescence resonance energy transfer (FRET) based molecular tension sensor for vinculin I demonstrated a spatial gradient of tension across vinculin in the direction of migration. To define this novel force-dependent role of vinculin in collective migration I took advantage of previously established shRNA based vinculin knock down Marin-Darby Canine Kidney (MDCK) epithelial cells.

The first part of my thesis comprises of my work demonstrating the mechanosensitive role of vinculin at AJ’s in collectively migrating cells. Using vinculin knockdown cells and vinculin mutants, which specifically disrupt vinculin’s ability to bind actin (VinI997A) or disrupt its ability to localize to AJs without affecting its localization at FAs (VinY822F), I establish a role of force across vinculin in E-cadherin internalization and clipping. Furthermore by measuring E-cadherin dynamics using fluorescence recovery after bleaching (FRAP) analysis I show that vinculin inhibition affects the turnover of E-cadherin at AJs. Together these data reveal a novel mechanosensitive role of vinculin in E-cadherin internalization and turnover in a migrating cell layer, which is contrary to the previously identified role of vinculin in potentiating E-cadherin junctions in a static monolayer.

For the last part of my thesis I designed a novel tension sensor to probe tension across N-cadherin (NTS). N-cadherin plays a critical role in cardiomyocytes, vascular smooth muscle cells, neurons and neural crest cells. Similar to E-cadherin, N-cadherin is also believed to bear tension and play a role in mechanotransduction pathways. To identify the role of tension across N-cadherin I designed a novel FRET-based molecular tension sensor for N-cadherin. I tested the ability of NTS to sense molecular tension in vascular smooth muscle cells, cardiomyocytes and cancer cells. Finally in collaboration with the Horwitz lab we have been able to show a role of tension across N-cadherin in synaptogenesis of neurons.