Characterisation of the vibrio cholerae antibiotic resistance var operon

The discovery and use of antibiotics in the chemotherapy of bacterial infections has revolutionised medicine as it is today. Unfortunately, the progressive use of antibiotics has promoted the evolution of bacterial defences against these mediators and thus the emergence of antibiotic resistance. Multidrug resistance (MDR) in bacterial pathogens has grown with such rapid progression that it now threatens to compromise the effective chemotherapy of a plethora of diseases. This thesis aspires to elucidate the molecular resistance mechanisms adopted by these bacteria, in order to expand our knowledge and to assist in the development of new therapeutic approaches to circumvent these mechanisms. On this basis, this thesis presents insights into a novel Vibrio cholerae antibiotic resistance, var, operon that encodes a metallo- β -lactamase (Mßl), VarG, and a tripartite ATP-binding cassette-type (ABC-type) transport system, VarACDEF that has substrate specificities for antimicrobial peptides and macrolide antibiotics. Mßls are fast emerging as a primary resistance mechanism, possibly as a consequence of the introduction of newer ß-lactam antibiotics such as the carbapenems in response to increasing Gram-negative bacterial resistance. Fascinatingly, the ABC transporter, through secondary structure predictions, has been envisaged to adopt a tripartite structure similar to the MDR transporter, AcrAB-TolC, from the resistance nodulation and cell division (RND) family. The structural characterisation of this system would be the first such tripartite system to be elucidated and may bring new insights into how Gram-negative bacteria may have evolved to tackle the issue that threatens its existence. The resistance mechanisms in the var Operon are believed to be under the control of a LysR-type transcriptional regulatory protein (LTTR), VarR. LTTR proteins form one of the largest transcriptional regulatory families with extremely diverse functions ranging from amino acid biosynthesis to CO(_2) fixation. VarR binds to three distinct promoter regions, varRG, varGA and varBC located upstream and adjacent to VarG, VarA an AcrA-like membrane fusion protein and VarC a TolC-like outer membrane protein, respectively. VarR has also been shown to act as a repressor at the varRG promoter region in the absence of its substrate. Interestingly, the mechanism of regulation by VarR is strikingly similar to the well documented LTTR, AmpR and serine ß-lactamase AmpC system that are found in many pathogenic bacteria. It could be that V. cholerae has evolved from this regular system and developed a ß-lactamase that would prove more beneficial in light of current selective pressures. Contrary to LTTRs being notoriously recalcitrant to purification due to their low solubility, this thesis reports the successful purification and crystallisation of full-length VarR in the presence and absence of its cognate promoter DNA. Elucidating the structural characteristics of VarR would be the first such regulator associated with MDR in the LTTR family. This would advance the knowledge on the only currently existing full-length crystal structure of a LTTR, CbnR, and will provide further insights into how structural conformations may lead to dissociation from the promoter and induction of gene expression. Understanding the mechanism by which VarR induces expression of these resistance mechanisms is paramount for future strategies to prevent the emergence of MDR microorganisms. Although these mechanisms of MDR maybe elucidated in V. cholerae, the evolutionary relatedness and conservation of structure and function in all families will enable this information to be related to similar systems in alternative bacterial species

Defining endogenous TACC3–chTOG–clathrin–GTSE1 interactions at the mitotic spindle using induced relocalization

A multiprotein complex containing TACC3, clathrin and other proteins has been implicated in mitotic spindle stability. To disrupt this complex in an anti-cancer context, we need to understand its composition and how it interacts with microtubules. Induced relocalization of proteins in cells is a powerful way to analyze protein–protein interactions and, additionally, monitor where and when these interactions occur. We used CRISPR/Cas9 gene editing to add tandem FKBP–GFP tags to each complex member. The relocalization of endogenous tagged protein from the mitotic spindle to mitochondria and assessment of the effect on other proteins allowed us to establish that TACC3 and clathrin are core complex members and that chTOG (also known as CKAP5) and GTSE1 are ancillary to the complex, binding respectively to TACC3 and clathrin, but not each other. We also show that PIK3C2A, a clathrin-binding protein that was proposed to stabilize the TACC3–chTOG–clathrin–GTSE1 complex during mitosis, is not a member of the complex. This work establishes that targeting the TACC3–clathrin interface or their microtubule-binding sites are the two strategies most likely to disrupt spindle stability mediated by this multiprotein complex

The Vibrio cholerae var regulon encodes a metallo-β-lactamase and an antibiotic efflux pump, which are regulated by VarR, a LysR-type transcription factor

The genome sequence of V. cholerae O1 Biovar Eltor strain N16961 has revealed a putative antibiotic resistance (var) regulon that is predicted to encode a transcriptional activator (VarR), which is divergently transcribed relative to the putative resistance genes for both a metallo-β-lactamase (VarG) and an antibiotic efflux-pump (VarABCDEF). We sought to test whether these genes could confer antibiotic resistance and are organised as a regulon under the control of VarR. VarG was overexpressed and purified and shown to have β-lactamase activity against penicillins, cephalosporins and carbapenems, having the highest activity against meropenem. The expression of VarABCDEF in the Escherichia coli (ΔacrAB) strain KAM3 conferred resistance to a range of drugs, but most significant resistance was to the macrolide spiramycin. A gel-shift analysis was used to determine if VarR bound to the promoter regions of the resistance genes. Consistent with the regulation of these resistance genes, VarR binds to three distinct intergenic regions, varRG, varGA and varBC located upstream and adjacent to varG, varA and varC, respectively. VarR can act as a repressor at the varRG promoter region; whilst this repression was relieved upon addition of β-lactams, these did not dissociate the VarR/varRG-DNA complex, indicating that the de-repression of varR by β-lactams is indirect. Considering that the genomic arrangement of VarR-VarG is strikingly similar to that of AmpR-AmpC system, it is possible that V. cholerae has evolved a system for resistance to the newer β-lactams that would prove more beneficial to the bacterium in light of current selective pressures

Measurement of inclusive D*+- and associated dijet cross sections in photoproduction at HERA

Inclusive photoproduction of D*+- mesons has been measured for photon-proton centre-of-mass energies in the range 130 < W < 280 GeV and a photon virtuality Q^2 < 1 GeV^2. The data sample used corresponds to an integrated luminosity of 37 pb^-1. Total and differential cross sections as functions of the D* transverse momentum and pseudorapidity are presented in restricted kinematical regions and the data are compared with next-to-leading order (NLO) perturbative QCD calculations using the "massive charm" and "massless charm" schemes. The measured cross sections are generally above the NLO calculations, in particular in the forward (proton) direction. The large data sample also allows the study of dijet production associated with charm. A significant resolved as well as a direct photon component contribute to the cross section. Leading order QCD Monte Carlo calculations indicate that the resolved contribution arises from a significant charm component in the photon. A massive charm NLO parton level calculation yields lower cross sections compared to the measured results in a kinematic region where the resolved photon contribution is significant.Comment: 32 pages including 6 figure

Plastisol Foaming Process. Decomposition of the Foaming Agent, Polymer Behavior in the Corresponding Temperature Range and Resulting Foam Properties

The decomposition of azodicarbonamide, used as foaming agent in PVC - plasticizer (1/1) plastisols was studied by DSC. Nineteen different plasticizers, all belonging to the ester family, two being polymeric (polyadipates), were compared. The temperature of maximum decomposition rate (in anisothermal regime at 5 K min-1 scanning rate), ranges between 434 and 452 K. The heat of decomposition ranges between 8.7 and 12.5 J g -1. Some trends of variation of these parameters appear significant and are discussed in terms of solvent (matrix) and viscosity effects on the decomposition reactions. The shear modulus at 1 Hz frequency was determined at the temperature of maximum rate of foaming agent decomposition, and differs significantly from a sample to another. The foam density was determined at ambient temperature and the volume fraction of bubbles was used as criterion to judge the efficiency of the foaming process. The results reveal the existence of an optimal shear modulus of the order of 2 kPa that corresponds roughly to plasticizer molar masses of the order of 450 ± 50 g mol-1. Heavier plasticizers, especially polymeric ones are too difficult to deform. Lighter plasticizers such as diethyl phthalate (DEP) deform too easily and presumably facilitate bubble collapse

D* Production in Deep Inelastic Scattering at HERA

This paper presents measurements of D^{*\pm} production in deep inelastic scattering from collisions between 27.5 GeV positrons and 820 GeV protons. The data have been taken with the ZEUS detector at HERA. The decay channel $D^{*+}\to (D^0 \to K^- \pi^+) \pi^+$ (+ c.c.) has been used in the study. The $e^+p$ cross section for inclusive D^{*\pm} production with $5<Q^2<100 GeV^2$ and $y<0.7$ is 5.3 \pms 1.0 \pms 0.8 nb in the kinematic region {$1.3<p_T(D^{*\pm})<9.0$ GeV and $| \eta(D^{*\pm}) |<1.5$}. Differential cross sections as functions of p_T(D^{*\pm}), $\eta(D^{*\pm}), W$ and $Q^2$ are compared with next-to-leading order QCD calculations based on the photon-gluon fusion production mechanism. After an extrapolation of the cross section to the full kinematic region in p_T(D^{*\pm}) and $\eta$(D^{*\pm}), the charm contribution $F_2^{c\bar{c}}(x,Q^2)$ to the proton structure function is determined for Bjorken $x$ between 2 $\cdot$ 10$^{-4}$ and 5 $\cdot$ 10$^{-3}$.Comment: 17 pages including 4 figure

Observation of Scaling Violations in Scaled Momentum Distributions at HERA

Charged particle production has been measured in deep inelastic scattering (DIS) events over a large range of $x$ and $Q^2$ using the ZEUS detector. The evolution of the scaled momentum, $x_p$, with $Q^2,$ in the range 10 to 1280 $GeV^2$, has been investigated in the current fragmentation region of the Breit frame. The results show clear evidence, in a single experiment, for scaling violations in scaled momenta as a function of $Q^2$.Comment: 21 pages including 4 figures, to be published in Physics Letters B. Two references adde

Observation of hard scattering in photoproduction events with a large rapidity gap at HERA

Events with a large rapidity gap and total transverse energy greater than 5 GeV have been observed in quasi-real photoproduction at HERA with the ZEUS detector. The distribution of these events as a function of the $\gamma p$ centre of mass energy is consistent with diffractive scattering. For total transverse energies above 12 GeV, the hadronic final states show predominantly a two-jet structure with each jet having a transverse energy greater than 4 GeV. For the two-jet events, little energy flow is found outside the jets. This observation is consistent with the hard scattering of a quasi-real photon with a colourless object in the proton.Comment: 19 pages, latex, 4 figures appended as uuencoded fil

Measurement of Jet Shapes in Photoproduction at HERA

The shape of jets produced in quasi-real photon-proton collisions at centre-of-mass energies in the range $134-277$ GeV has been measured using the hadronic energy flow. The measurement was done with the ZEUS detector at HERA. Jets are identified using a cone algorithm in the $\eta - \phi$ plane with a cone radius of one unit. Measured jet shapes both in inclusive jet and dijet production with transverse energies $E^{jet}_T>14$ GeV are presented. The jet shape broadens as the jet pseudorapidity ($\eta^{jet}$) increases and narrows as $E^{jet}_T$ increases. In dijet photoproduction, the jet shapes have been measured separately for samples dominated by resolved and by direct processes. Leading-logarithm parton-shower Monte Carlo calculations of resolved and direct processes describe well the measured jet shapes except for the inclusive production of jets with high $\eta^{jet}$ and low $E^{jet}_T$. The observed broadening of the jet shape as $\eta^{jet}$ increases is consistent with the predicted increase in the fraction of final state gluon jets.Comment: 29 pages including 9 figure

Measurement of the diffractive structure function in deep inelastic scattering at HERA

This paper presents an analysis of the inclusive properties of diffractive deep inelastic scattering events produced in $ep$ interactions at HERA. The events are characterised by a rapidity gap between the outgoing proton system and the remaining hadronic system. Inclusive distributions are presented and compared with Monte Carlo models for diffractive processes. The data are consistent with models where the pomeron structure function has a hard and a soft contribution. The diffractive structure function is measured as a function of \xpom, the momentum fraction lost by the proton, of $\beta$, the momentum fraction of the struck quark with respect to \xpom, and of $Q^2$. The \xpom dependence is consistent with the form \xpoma where $a~=~1.30~\pm~0.08~(stat)~^{+~0.08}_{-~0.14}~(sys)$ in all bins of $\beta$ and $Q^2$. In the measured $Q^2$ range, the diffractive structure function approximately scales with $Q^2$ at fixed $\beta$. In an Ingelman-Schlein type model, where commonly used pomeron flux factor normalisations are assumed, it is found that the quarks within the pomeron do not saturate the momentum sum rule.Comment: 36 pages, latex, 11 figures appended as uuencoded fil