Molecular analysis of interactions between normal and transformed epithelial cells at early stages of cancerogenesis

Carcinomas begin with a single transformed cell in an otherwise normal epithelial monolayer. To study the nature of interactions between a transformed cell and its neighbours at this initial stage of tumourigenesis a tetracycline-inducible system driving expression of a constitutively active form of oncogenic Ras (RasV12) was established in MDCK epithelial cells by the Fujita laboratory. Upon interaction with normal cells, RasV12 cells most commonly undergo apical extrusion from an epithelial monolayer. In order to identify proteins and pathways involved in interactions between normal and transformed cells, I have performed several biochemical screens described in this thesis. Firstly, I have shown that Hsp90β, identified previously in a 2D gel screen, is increased in RasV12 cells surrounded by normal neighbours in a non-cell-autonomous fashion. By using inhibitors and a dominant negative form of Hsp90, I have shown that upregulation of this chaperone is most likely a part of a stress response delaying extrusion of transformed cells. Secondly, I have performed a screen for tyrosine phosphorylated proteins and identified myosin IE and plectin as modified in mixed cultures of normal and transformed cells. Finally, I have undertaken quantitative mass spectrometry of phosphorylated peptides using SILAC labelling to assess changes in RasV12 cells upon their interaction with normal cells. In this screen I have found that an actin anticapping protein, VASP, is phosphorylated on serine 239 in RasV12 cells interacting with normal cells. This modification is known to inhibits its related to actin function. I have shown that depletion of VASP in RasV12 cells results in their enhanced extrusion from normal monolayers, most likely due to their compromised attachment. The phosphorylation on serine 239 may be an early step in extrusion, contributing to disassembly of focal adhesions and stress fibres in transformed cells. I have also studied the role of another protein identified in the SILAC screen, MRCKβ, and shown that its depletion results in enhanced extrusion of RasV12 cells from normal monolayers

Measuring health inequality among children in developing countries: does the choice of the indicator of economic status matter?

Background Currently, poor-rich inequalities in health in developing countries receive a lot of attention from both researchers and policy makers. Since measuring economic status in developing countries is often problematic, different indicators of wealth are used in different studies. Until now, there is a lack of evidence on the extent to which the use of different measures of economic status affects the observed magnitude of health inequalities. Methods This paper provides this empirical evidence for 10 developing countries, using the Demographic and Health Surveys data-set. We compared the World Bank asset index to three alternative wealth indices, all based on household assets. Under-5 mortality and measles immunisation coverage were the health outcomes studied. Poor-rich inequalities in under-5 mortality and measles immunisation coverage were measured using the Relative Index of Inequality. Results Comparing the World Bank index to the alternative indices, we found that (1) the relative position of households in the national wealth hierarchy varied to an important extent with the asset index used, (2) observed poor-rich inequalities in under-5 mortality and immunisation coverage often changed, in some cases to an important extent, and that (3) the size and direction of this change varied per country, index, and health indicator. Conclusion Researchers and policy makers should be aware that the choice of the measure of economic status influences the observed magnitude of health inequalities, and that differences in health inequalities between countries or time periods, may be an artefact of different wealth measures used

Nonperturbative Tests of Three-Dimensional Dualities

We test several conjectural dualities between strongly coupled superconformal field theories in three dimensions by computing their exact partition functions on a three-sphere as a function of Fayet-Iliopoulos and mass parameters. The calculation is carried out using localization of the path integral and the matrix model previously derived for superconformal N = 2 gauge theories. We verify that the partition functions of quiver theories related by mirror symmetry agree provided the mass parameters and the Fayet-Iliopoulos parameters are exchanged, as predicted. We carry out a similar calculation for the mirror of N = 8 super-Yang-Mills theory and show that its partition function agrees with that of the ABJM theory at unit Chern-Simons level. This provides a nonperturbative test of the conjectural equivalence of the two theories in the conformal limit

Supersymmetry enhancement by monopole operators

We describe a method which allows one to study hidden symmetries in a large class of strongly coupled supersymmetric gauge theories in three dimensions. We apply this method to the ABJM theory and to the infrared limit of N=4 SQCD with adjoint and fundamental matter. We show that the U(N) ABJM model with Chern-Simons level k=1 or k=2 has hidden N=8 supersymmetry. Hidden supersymmetry is also shown to occur in N=4 d=3 SQCD with one fundamental and one adjoint hypermultiplet. The latter theory, as well as the U(N) ABJM theory at k=1, are shown to have a decoupled free sector. This provides evidence that both models are dual to the infrared limit of N=8 U(N) super-Yang-Mills theory.Comment: 29 pages, late

NeuroQuantify -- An Image Analysis Software for Detection and Quantification of Neurons and Neurites using Deep Learning

The segmentation of cells and neurites in microscopy images of neuronal networks provides valuable quantitative information about neuron growth and neuronal differentiation, including the number of cells, neurites, neurite length and neurite orientation. This information is essential for assessing the development of neuronal networks in response to extracellular stimuli, which is useful for studying neuronal structures, for example, the study of neurodegenerative diseases and pharmaceuticals. However, automatic and accurate analysis of neuronal structures from phase contrast images has remained challenging. To address this, we have developed NeuroQuantify, an open-source software that uses deep learning to efficiently and quickly segment cells and neurites in phase contrast microscopy images. NeuroQuantify offers several key features: (i) automatic detection of cells and neurites; (ii) post-processing of the images for the quantitative neurite length measurement based on segmentation of phase contrast microscopy images, and (iii) identification of neurite orientations. The user-friendly NeuroQuantify software can be installed and freely downloaded from GitHub https://github.com/StanleyZ0528/neural-image-segmentation

Phases of planar 5-dimensional supersymmetric Chern-Simons theory

In this paper we investigate the large-$N$ behavior of 5-dimensional $\mathcal{N}=1$ super Yang-Mills with a level $k$ Chern-Simons term and an adjoint hypermultiplet. As in three-dimensional Chern-Simons theories, one must choose an integration contour to completely define the theory. Using localization, we reduce the path integral to a matrix model with a cubic action and compute its free energy in various scenarios. In the limit of infinite Yang-Mills coupling and for particular choices of the contours, we find that the free-energy scales as $N^{5/2}$ for $U(N)$ gauge groups with large values of the Chern-Simons 't\,Hooft coupling, $\tilde\lambda\equiv N/k$. If we also set the hypermultiplet mass to zero, then this limit is a superconformal fixed point and the $N^{5/2}$ behavior parallels other fixed points which have known supergravity duals. We also demonstrate that $SU(N)$ gauge groups cannot have this $N^{5/2}$ scaling for their free-energy. At finite Yang-Mills coupling we establish the existence of a third order phase transition where the theory crosses over from the Yang-Mills phase to the Chern-Simons phase. The phase transition exists for any value of $\tilde\lambda$, although the details differ between small and large values of $\tilde\lambda$. For pure Chern-Simons theories we present evidence for a chain of phase transitions as $\tilde\lambda$ is increased. We also find the expectation values for supersymmetric circular Wilson loops in these various scenarios and show that the Chern-Simons term leads to different physical properties for fundamental and anti-fundamental Wilson loops. Different choices of the integration contours also lead to different properties for the loops.Comment: 40 pages, 17 figures, Minor corrections, Published versio

Incident venous thromboembolic events in the Prospective Study of Pravastatin in the Elderly at Risk (PROSPER)

<p>Background: Venous thromboembolic events (VTE), including deep venous thrombosis and pulmonary embolism, are common in older age. It has been suggested that statins might reduce the risk of VTE however positive results from studies of middle aged subjects may not be generalisable to elderly people. We aimed to determine the effect of pravastatin on incident VTE in older people; we also studied the impact of clinical and plasma risk variables.</p> <p>Methods: This study was an analysis of incident VTE using data from the Prospective Study of Pravastatin in the Elderly at Risk (PROSPER), a randomized, double-blind, placebo-controlled trial of pravastatin in men and women aged 70-82. Mean follow-up was 3.2 years. Risk for VTE was examined in non-warfarin treated pravastatin (n = 2834) and placebo (n = 2865) patients using a Cox's proportional hazard model, and the impact of other risk factors assessed in a multivariate forward stepwise regression analysis. Baseline clinical characteristics, blood biochemistry and hematology variables, plasma levels of lipids and lipoproteins, and plasma markers of inflammation and adiposity were compared. Plasma markers of thrombosis and hemostasis were assessed in a nested case (n = 48) control (n = 93) study where the cohort was those participants, not on warfarin, for whom data were available.</p> <p>Results: There were 28 definite cases (1.0%) of incident VTE in the pravastatin group recipients and 20 cases (0.70%) in placebo recipients. Pravastatin did not reduce VTE in PROSPER compared to placebo [unadjusted hazard ratio (95% confidence interval) 1.42 (0.80, 2.52) p = 0.23]. Higher body mass index (BMI) [1.09 (1.02, 1.15) p = 0.0075], country [Scotland vs Netherlands 4.26 (1.00, 18.21) p = 0.050 and Ireland vs Netherlands 6.16 (1.46, 26.00) p = 0.013], lower systolic blood pressure [1.35 (1.03, 1.75) p = 0.027] and lower baseline Mini Mental State Examination (MMSE) score [1.19 (1.01, 1.41) p = 0.034] were associated with an increased risk of VTE, however only BMI, country and systolic blood pressure remained significant on multivariate analysis. In a nested case control study of definite VTE, plasma Factor VIII levels were associated with VTE [1.52 (1.01, 2.28), p = 0.044]. However no other measure of thrombosis and haemostasis was associated with increased risk of VTE.</p> <p>Conclusions: Pravastatin does not prevent VTE in elderly people at risk of vascular disease. Blood markers of haemostasis and inflammation are not strongly predictive of VTE in older age however BMI, country and lower systolic blood pressure are independently associated with VTE risk.</p&gt

A mathematical model for breath gas analysis of volatile organic compounds with special emphasis on acetone

Recommended standardized procedures for determining exhaled lower respiratory nitric oxide and nasal nitric oxide have been developed by task forces of the European Respiratory Society and the American Thoracic Society. These recommendations have paved the way for the measurement of nitric oxide to become a diagnostic tool for specific clinical applications. It would be desirable to develop similar guidelines for the sampling of other trace gases in exhaled breath, especially volatile organic compounds (VOCs) which reflect ongoing metabolism. The concentrations of water-soluble, blood-borne substances in exhaled breath are influenced by: (i) breathing patterns affecting gas exchange in the conducting airways; (ii) the concentrations in the tracheo-bronchial lining fluid; (iii) the alveolar and systemic concentrations of the compound. The classical Farhi equation takes only the alveolar concentrations into account. Real-time measurements of acetone in end-tidal breath under an ergometer challenge show characteristics which cannot be explained within the Farhi setting. Here we develop a compartment model that reliably captures these profiles and is capable of relating breath to the systemic concentrations of acetone. By comparison with experimental data it is inferred that the major part of variability in breath acetone concentrations (e.g., in response to moderate exercise or altered breathing patterns) can be attributed to airway gas exchange, with minimal changes of the underlying blood and tissue concentrations. Moreover, it is deduced that measured end-tidal breath concentrations of acetone determined during resting conditions and free breathing will be rather poor indicators for endogenous levels. Particularly, the current formulation includes the classical Farhi and the Scheid series inhomogeneity model as special limiting cases.Comment: 38 page

PKA-regulated VASP phosphorylation promotes extrusion of transformed cells from the epithelium.

At the early stages of carcinogenesis, transformation occurs in single cells within tissues. In an epithelial monolayer, such mutated cells are recognized by their normal neighbors and are often apically extruded. The apical extrusion requires cytoskeletal reorganization and changes in cell shape, but the molecular switches involved in the regulation of these processes are poorly understood. Here, using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry, we have identified proteins that are modulated in transformed cells upon their interaction with normal cells. Phosphorylation of VASP at serine 239 is specifically upregulated in Ras(V12)-transformed cells when they are surrounded by normal cells. VASP phosphorylation is required for the cell shape changes and apical extrusion of Ras-transformed cells. Furthermore, PKA is activated in Ras-transformed cells that are surrounded by normal cells, leading to VASP phosphorylation. These results indicate that the PKA-VASP pathway is a crucial regulator of tumor cell extrusion from the epithelium, and they shed light on the events occurring at the early stage of carcinogenesis

New Abundant Microbial Groups in Aquatic Hypersaline Environments

We describe the microbiota of two hypersaline saltern ponds, one of intermediate salinity (19%) and a NaCl saturated crystallizer pond (37%) using pyrosequencing. The analyses of these metagenomes (nearly 784 Mb) reaffirmed the vast dominance of Haloquadratum walsbyi but also revealed novel, abundant and previously unsuspected microbial groups. We describe for the first time, a group of low GC Actinobacteria, related to freshwater Actinobacteria, abundant in low and intermediate salinities. Metagenomic assembly revealed three new abundant microbes: a low-GC euryarchaeon with the lowest GC content described for any euryarchaeon, a high-GC euryarchaeon and a gammaproteobacterium related to Alkalilimnicola and Nitrococcus. Multiple displacement amplification and sequencing of the genome from a single archaeal cell of the new low GC euryarchaeon suggest a photoheterotrophic and polysaccharide-degrading lifestyle and its relatedness to the recently described lineage of Nanohaloarchaea. These discoveries reveal the combined power of an unbiased metagenomic and single cell genomic approach