Utilising Uracil DNA Glycosylase to detect the presence of 5-Methylcytosine

DNA is regularly subjected to endogenous and exogenous reagents that cause mutations that can be detrimental to a cell if they are not repaired. One class of enzymes responsible for DNA repair is the family of DNA glycosylases and their role is to remove damaged bases. Uracil DNA Glycosylase (UDG) is a member of this family and is highly specific, removing only uracil, an RNA base, from DNA. Uracil arises in DNA through misincorporation of deoxyuridine monophosphate (dUMP) creating an A.U base pair, or through deamination of cytosine resulting in a G.U base pair. Though UDG acts on A.U pairs, this is not it’s primarily role as A.U pairings are not mutagenic. However the G.U mispair is highly mutagenic and leads to a G.C to A.T transition on subsequent rounds of replication. UDG only reacts with uracil and has no activity at thymine since the 5-methyl group on the base is excluded from the active site. This thesis examines mutants of UDG that can cleave cytosine but not 5-methylcytosine. Methylation of cytosine at CpG sites leads to gene silencing and is an important epigenetic signal. Knowing the methylation state of cytosines will therefore be important for understanding gene control and may be beneficial for treating many diseases. The most common method for detecting cytosine methylation uses a bisulphite reaction followed by normal DNA sequencing methods. This process has several drawbacks and the aim of this work is to create an enzyme that is capable of distinguishing between5-methylcytosine and cytosine. It has been reported that mutation of a critical asparagine in UDG to an aspartate allows the enzyme to accommodate cytosine into its active site; generating a cytosine DNA glycosylase (CDG). Using the natural ability of UDG to distinguish between uracil and thymine due to the presence of the 5-methyl group, we hypothesised that the mutant enzyme should be able to discriminate between5-methylcytosine and cytosine, which differ by the presence or absence of a methyl group in the same position. E. coli and human CDGs were prepared and their ability to remove cytosine or 5-methylcytosine examined when placed in different sequence contexts. hCDG was generated through complete gene synthesis of hUDG followed by the N204D mutation. The corresponding mutation in E.coli (N123D) generates a highly cytotoxic enzyme that cannot even be cloned in pUC19. As L191 aids base flipping, mutation to alanine (L191A) renders the enzyme inactive; activity can then be rescued using a bulky synthetic nucleoside that occupies the base pair and forces the target base into an extrahelical conformation. The L191A mutation was followed by N123D to generate an expressible and functional eCDG, denoted eCYDG. We demonstrate that these mutants have cytosine glycosylase activity when the cytosine is mispaired or unpaired, but not when paired with guanine, and show no activity against5-methylcytosine in any context. The activity of these CDGs varies with the stability of the base pair, with the fastest cleavage rates being obtained with the least stable base pairs, and also varies with the local sequence context. As CDGs are able to discriminate between cytosine and 5-methylcytosine we began development of a real-time PCR assay for detection of 5-methylcytosine. This employed a hexaethylene glycol (HEG) linker opposite the target cytosine, as this produces one of the fastest cleavage rates and cannot be read by a DNA polymerase

Involvement of a putative intercellular signal-recognizing G protein-coupled receptor in the engulfment of Salmonella by the protozoan Tetrahymena

In an effort to investigate the molecular basis of protozoa engulfment-mediated hypervirulence of Salmonella in cattle, we evaluated protozoan G protein-coupled receptors (GPCRs) as transducers of Salmonella engulfment by the model protozoanTetrahymena. Our laboratory previously demonstrated that non-pathogenic protozoa (including Tetrahymena) engulf Salmonella and then exacerbate its virulence in cattle, but the mechanistic details of the phenomenon are not fully understood. GPCRs were investigated since these receptors facilitate phagocytosis of particulates byTetrahymena, and a GPCR apparently modulates bacterial engulfment for the pathogenic protozoan Entamoeba histolytica. A database search identified three putative Tetrahymena GPCRs, based on sequence homologies and predicted transmembrane domains, that were the focus of this study. Salmonella engulfment by Tetrahymenawas assessed in the presence of suramin, a non-specific GPCR inhibitor. Salmonella engulfment was also assessed in Tetrahymena in which expression of putative GPCRs was knocked-down using RNAi. A candidate GPCR was then expressed in a heterologous yeast expression system for further characterization. Our results revealed that Tetrahymena were less efficient at engulfing Salmonella in the presence of suramin. Engulfment was reduced concordantly with a reduction in the density of protozoa. RNAi-based studies revealed that knock-down of one the Tetrahymena GPCRs caused diminished engulfment of Salmonella. Tetrahymena lysates activated this receptor in the heterologous expression system. These data demonstrate that the Tetrahymena receptor is a putative GPCR that facilitates bacterial engulfment by Tetrahymena. Activation of the putative GPCR seemed to be related to protozoan cell density, suggesting that its cognate ligand is an intercellular signaling molecule

Cluster size distributions in particle systems with asymmetric dynamics

We present exact and asymptotic results for clusters in the one-dimensional totally asymmetric exclusion process (TASEP) with two different dynamics. The expected length of the largest cluster is shown to diverge logarithmically with increasing system size for ordinary TASEP dynamics and as a logarithm divided by a double logarithm for generalized dynamics, where the hopping probability of a particle depends on the size of the cluster it belongs to. The connection with the asymptotic theory of extreme order statistics is discussed in detail. We also consider a related model of interface growth, where the deposited particles are allowed to relax to the local gravitational minimum.Comment: 12 pages, 3 figures, RevTe

Assessing Dysplasia of a Bronchial Biopsy with FTIR Spectroscopic Imaging

An FTIR image of an 8 µm section of de-paraffinised bronchial biopsy that shows a histological transition from normal to severe dysplasia/squamous cell carcinoma (SCC) insitu was obtained in transmission by stitching together images of 256 x 256 µm recorded using a 96 x 96 element FPA detector. Each pixel spectrum was calculated from 128 co-added interferograms at 4 cm−1 resolution. In order to improve the signal to noise ratio, blocks of 4x4 adjacent pixels were subsequently averaged. Analyses of this spectral image, after conversion of the spectra to their second derivatives, show that the epithelium and the lamina propria tissue types can be distinguished using the area of troughs at either 1591, 1334, 1275 or 1215 cm−1 or, more effectively, by separation into two groups by hierarchical clustering (HCA) of the 1614-1465 region. Due to an insufficient signal to noise ratio, disease stages within the image could not be distinguished with this extent of pixel averaging. However, after separation of the cell types, disease stages within either the epithelium or the lamina propria could be distinguished if spectra were averaged from larger, manually selected areas of the tissue. Both cell types reveal spectral differences that follow a transition from normal to cancerous histology. For example, spectral changes that occurred in the epithelium over the transition from normal to carcinoma insitu could be seen in the 1200-1000 cm−1 region, particularly as a decrease in the second derivative troughs at 1074 and 1036 cm−1 , consistent with changes in some form of carbohydrate. Spectral differences that indicate a disease transition from normal to carcinoma in the lamina propria could be seen in the 1350-1175 cm−1 and 1125-1030 cm−1 regions. Thus demonstrating that a progression from healthy to severe dysplasia/squamous cell carcinoma (SCC) insitu can be seen using FTIR spectroscopic imaging and multivariate analysis

Nuclear charm and bottom production: a comparison among high energy approaches

We calculate the nucleon and nuclear photoproduction cross sections for heavy quarks within the $k_{\perp}$-factorization formalism, considering the current high energy approaches which include nuclear and saturation effects. Our results demonstrate that a future experimental analysis of this process would allow to constraint the QCD dynamics at high energies.Comment: 10 pages, 2 figures. Version to be published in Eur. Phys. J.

Characterisation of Osteopontin in an In Vitro Model of Embryo Implantation

At the onset of pregnancy, embryo implantation is initiated by interactions between the endometrial epithelium and the outer trophectoderm cells of the blastocyst. Osteopontin (OPN) is expressed in the endometrium and is implicated in attachment and signalling roles at the embryo–epithelium interface. We have characterised OPN in the human endometrial epithelial Ishikawa cell line using three different monoclonal antibodies, revealing at least nine distinct molecular weight forms and a novel secretory pathway localisation in the apical domain induced by cell organisation into a confluent epithelial layer. Mouse blastocysts co-cultured with Ishikawa cell layers served to model embryo apposition, attachment and initial invasion at implantation. Exogenous OPN attenuated initial, weak embryo attachment to Ishikawa cells but did not affect the attainment of stable attachment. Notably, exogenous OPN inhibited embryonic invasion of the underlying cell layer, and this corresponded with altered expression of transcription factors associated with differentiation from trophectoderm (Gata2) to invasive trophoblast giant cells (Hand1). These data demonstrate the complexity of endometrial OPN forms and suggest that OPN regulates embryonic invasion at implantation by signalling to the trophectoder

Prompt photon and associated heavy quark production at hadron colliders with kt-factorization

In the framework of the kt-factorization approach, the production of prompt photons in association with a heavy (charm or beauty) quarks at high energies is studied. The consideration is based on the O(\alpha \alpha_s^2) off-shell amplitudes of gluon-gluon fusion and quark-(anti)quark interaction subprocesses. The unintegrated parton densities in a proton are determined using the Kimber-Martin-Ryskin prescription. The analysis covers the total and differential cross sections and extends to specific angular correlations between the produced prompt photons and muons originating from the semileptonic decays of associated heavy quarks. Theoretical uncertainties of our evaluations are studied and comparison with the results of standard NLO pQCD calculations is performed. Our numerical predictions are compared with the recent experimental data taken by the D0 and CDF collaborations at the Tevatron. Finally, we extend our results to LHC energies.Comment: 14 pages, 10 figure

Osmotic stress induces JNK-dependent embryo invasion in a model of implantation

In vitro culture during assisted reproduction technologies (ARTs) exposes pre-implantation embryos to environmental stressors, such as non-physiological nutritional, oxidative and osmotic conditions. The effects on subsequent implantation are not well understood but could contribute to poor ART efficiency and outcomes. We have used exposure to hyperosmolarity to investigate the effects of stress on the ability of embryos to interact with endometrial cells in an in vitro model. Culturing mouse blastocysts for 2 h in medium with osmolarity raised by 400 mosmol induced blastocoel collapse and re-expansion, but did not affect subsequent attachment to, or invasion of, the endometrial epithelial Ishikawa cell line. Inhibition of stress-responsive c-Jun N-terminal kinase (JNK) activity with SP600125 did not affect the intercellular interactions between these embryos and the epithelial cells. Four successive cycles of hyperosmotic stress at E5.5 had no effect on attachment, but promoted embryonic breaching of the epithelial cell layer by trophoblast giant cells in a JNK-dependent manner. These findings suggest that acute stress at the blastocyst stage may promote trophoblast breaching of the endometrial epithelium at implantation and implicates stress signalling through JNK in the process of trophectoderm differentiation into the invasive trophoblast necessary for the establishment of pregnancy. The data may lead to increased understanding of factors governing ART success rates and safety

Precise Stellar Radial Velocities of an M Dwarf with a Michelson Interferometer and a Medium-resolution Near-infrared Spectrograph

Precise near-infrared radial velocimetry enables efficient detection and transit verification of low-mass extrasolar planets orbiting M dwarf hosts, which are faint for visible-wavelength radial velocity surveys. The TripleSpec Exoplanet Discovery Instrument, or TEDI, is the combination of a variable-delay Michelson interferometer and a medium-resolution (R=2700) near-infrared spectrograph on the Palomar 200" Hale Telescope. We used TEDI to monitor GJ 699, a nearby mid-M dwarf, over 11 nights spread across 3 months. Analysis of 106 independent observations reveals a root-mean-square precision of less than 37 m/s for 5 minutes of integration time. This performance is within a factor of 2 of our expected photon-limited precision. We further decompose the residuals into a 33 m/s white noise component, and a 15 m/s systematic noise component, which we identify as likely due to contamination by telluric absorption lines. With further development this technique holds promise for broad implementation on medium-resolution near-infrared spectrographs to search for low-mass exoplanets orbiting M dwarfs, and to verify low-mass transit candidates.Comment: 55 pages and 13 figures in aastex format. Accepted by PAS