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Biochemical Characterization of Hydroxyproline-rich Glycoproteins in the Arabidopsis Root Cell WallChen, Yuning January 2012 (has links)
No description available.
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Expanding The Horizon Of Mycobacterial Stress Response : Discovery Of A Second (P)PPGPP Synthetase In Mycobacterium SmegmatisMurdeshwar, Maya S 09 1900 (has links) (PDF)
The stringent response is a highly conserved physiological response mounted by bacteria under stress (Ojha and Chatterji, 2001; Magnusson et al., 2005; Srivatsan and Wang, 2007; Potrykus and Cashel, 2008). Until recently, the only known players in this pathway were the (p)ppGpp synthesizing and hydrolyzing long RSH enzymes (Mittenhuber, 2001; Atkinson et al., 2011) - RelA and SpoT in Gram negative bacteria and the bifunctional Rel in Gram positive bacteria including mycobacteria. The existence of Short Alarmone Synthetases (SAS) (Lemos et al., 2007, Nanamiya et al., 2008; Das et al., 2009; Atkinson et al., 2011) and Short Alarmone Hydrolases (SAH) (Sun et al., 2010, Atkinson et al., 2011), small proteins possessing a single functional (p)ppGpp synthetase or hydrolase domain respectively, is a recent discovery that has modified this paradigm. Around the same time that the presence of the SAS proteins was reported, we chanced upon such small (p)ppGpp synthetases in the genus Mycobacterium. The stringent response in the soil saprophyte Mycobacterium smegmatis was first reported by Ojha and co-workers (Ojha et al., 2000), and the bifunctional RSH, RelMsm, responsible for mounting the stringent response in this bacterium, has been characterized in detail (Jain et al., 2006 and 2007). RelMsm was the only known RSH enzyme present in M. smegmatis, and consequently, a strain of M. smegmatis deleted for the relMsm gene (ΔrelMsm) (Mathew et al., 2004), was expected to show a null phenotype for (p)ppGpp production. In this body of work, we report the surprising observation that the M. smegmatis ΔrelMsm strain is capable of synthesizing (p)ppGpp in vivo. This unexpected turn of events led us to the discovery of a second (p)ppGpp synthetase in this bacterium. The novel protein was found to possess two functional domains – an RNase HII domain at the amino-terminus, and a (p)ppGpp synthetase or RSD domain at the carboxy-terminus. We have therefore named this protein ‘MS_RHII-RSD’, indicating the two activities present and identifying the organism from which it is isolated. Orthologs of this novel SAS protein occur in other species of mycobacteria, both pathogenic and non-pathogenic. In this study, we report the cloning, purification and in-depth functional characterization of MS_RHII-RSD, and speculate on its in vivo role in M. smegmatis.
Chapter 1 reviews the available literature in the field of stringent response research and lays the background to this study. A historical perspective is provided,
starting with the discovery of the stringent response in bacteria in the early 1960s, highlighting the development in this area till date. The roles played by the long and short RSH enzymes, ‘Magic Spot’ (p)ppGpp, the RNA polymerase enzyme complex, and a few other RNA and proteins are described, briefly outlining the inferences drawn from recent global gene expression and proteomics studies. The chapter concludes with a description of the motivation behind, and the scope of the present study.
Chapter 2 discusses the in vivo and in silico identification of MS_RHII-RSD in M. smegmatis. Experiments performed for the genotypic and phenotypic revalidation of M. smegmatis ΔrelMsm strain are described. Detailed bioinformatics analyses are provided for the in silico characterization of MS_RHII-RSD in terms of its domain architecture, in vivo localization, and protein structure prediction. A comprehensive list of the mycobacterial orthologs of MS_RHII-RSD from a few representative species of infectious and non-infectious mycobacteria is included.
Chapter 3 summarizes the materials and methods used in the cloning, purification, and the biophysical and biochemical characterization of full length MS_RHII-RSD and its two domain variants – RHII and RSD, respectively. A detailed description of the purification protocols highlighting the specific modifications and changes made is given. Peptide mass fingerprinting to confirm protein identity, as well as preliminary mass spectrometric, chromatographic, and circular dichroism-based characterization of the proteins under study is also provided.
Chapter 4 deals in detail with the in vivo and in vitro functional characterization of the RNase HII and (p)ppGpp synthesis activities of full length MS_RHII-RSD and its two domain variants - RHII and RSD, respectively. The RNase HII activity is characterized in vivo on the basis of a complementation assay in an E. coli strain deleted for the RNase H genes; while in vitro characterization is done by performing a FRET-based assay to monitor the degradation of a RNA•DNA hybrid substrate in vitro. The (p)ppGpp synthesis activity is characterized in terms of the substrate specificity, magnesium ion utilization, and a detailed analysis of the kinetic parameters involved. A comparison of the (p)ppGpp synthesis activity of MS_RHII-RSD vis-à-vis that of the classical RSH protein, RelMsm, is also provided. Inferences drawn from (p)ppGpp hydrolysis assays and the in vivo expression profile of MS_RHII-RSD in M. smegmatis wild type and ΔrelMsm strains are discussed. Based on the results of these functional assays, a model is proposed suggesting the probable in vivo role played by MS_RHII-RSD in M. smegmatis.
Chapter 5 describes the attempts at generating MS_RHII-RSD overexpression and knockout strains in M. smegmatis, using pJAM2-based mycobacterial expression system, and mycobacteriophage-based specialized transduction strategy, respectively. The detailed methodology and the principle behind the techniques used are explained. The results obtained so far, and the future work and strain characterization to be carried out in this respect are discussed.
Chapter 6 takes a slightly different route and summarizes the work carried out in characterizing the glycopeptidolipids (GPLs) from M. smegmatis biofilm cultures. A general introduction about the mycobacterial cell wall components, with special emphasis on GPLs, is provided. The detailed protocols for chemical composition and chromatographic analyses are mentioned, and the future scope of this work is discussed.
Appendix-1 briefly revisits the preliminary studies performed to determine the pppGpp binding site on M. smegmatis RNA polymerase using a mass spectrometry-based approach. Appendices-2, 3, 4 and 5 give a comprehensive list of the bacterial strains; PCR primers; antibiotics, buffers and media used; and the plasmid and phasmid maps, respectively.
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Natriuretic peptide receptor-C activation regulates vascular smooth muscle cell hypertrophy : molecular mechanismsJain, Ashish 12 1900 (has links)
Thesis evaluated by: Dr. Rémy Sauvé, Dr. Puttaswamy Manjunath and Dr. Madhu Anand-Srivastava. Thank you for all your help. / L’hypertension est associée au remodelage vasculaire dû à l’hyperprolifération et l’hypertrophie des cellules musculaires lisses vasculaires (CMLVs). Nous avons démontré par le passé l’implication de l’expression élevée des protéines Gqα et PLCβ1 dans les CMLVs de rats spontanément hypertendus (RSH) âgés de 16 semaines. Le C-ANP4-23 est un agoniste du récepteur au peptide natriurétique de type C (NPR-C) qui possède la capacité d’inhiber la synthèse de protéines en réponse aux peptides vasoactifs dans les CMLVs. Cette étude a eu pour but d’examiner si le C-ANP4-23 pouvait atténuer l’hypertrophie dans un modèle de rat souffrant d’hypertrophie cardiaque et d’explorer les mécanismes responsables de cette inhibition. Pour ce faire, des CMLVs aortiques de RSH âgés de 16 semaines ont été utilisées. Le taux de synthèse de protéines, un marqueur d’hypertrophie, a été déterminé par l’incorporation de (3H)leucine et l’expression des protéines a été déterminée par la technique d’immunobuvardage de type Western. Le volume cellulaire a été estimé par imagerie confocale tridimensionnelle. Le taux de synthèse de protéines et le volume cellulaire étaient considérablement accrus dans les CMLVs des RSH comparativement aux rats WKY et ont été largement atténués par le traitement au C-ANP4-23. De plus, le traitement au C-ANP4-23 a normalisé l’expression élevée du récepteur AT1 et des protéines Gqα et PLCβ1, des niveaux intracellulaires d’anions superoxide (O2-), de l’activité de la NADPH (de l’anglais nicotinamide adenine dinucleotide phosphate) oxydase, ainsi que l’expression des protéines Nox4 et de p47phox dans les CMLVs des RSH. En outre, le C-ANP4-23 a réduit l’activation des récepteurs à L’EGF (de l’anglais epidermal growth factor), au PDGF (de l’anglais platelet-derived growth factor), et à l’IGF-1 (de l’anglais insulin-like growth factor 1). Le C-ANP4-23 a également atténué la phosphorylation des ERK1/2 (de l’anglais extracellular regulated kinase1/2), AKT et c-Src. Ces résultats indiquent que l’activation du NPR-C par C-ANP4-23 a atténué l’hypertrophie des CMLVs par sa capacité à diminuer la surexpression du récepteur AT1, l’expression élevée des protéines Gqα/PLCβ1, le stress oxydatif accru, l’activation augmentée des facteurs de croissance et l’augmentation de la phosphorylation des voies de signalisation MAPK/AKT. Ainsi, ces travaux suggèrent que le C-ANP4-23 peut être utilisé comme agent thérapeutique pour le traitement des complications vasculaires associées à l’hypertension et à l’athérosclérose. / Hypertension is associated with vascular remodelling due to hyperproliferation
and hypertrophy of vascular smooth muscle cells (VSMCs). We earlier showed the
implication of enhanced expression of Gqα and PLCβ1 proteins in VSMCs from 16-
week-old spontaneously hypertensive rats (SHR). The present study was undertaken to
investigate whether C-ANP4-23, a natriuretic peptide receptor-C (NPR-C) agonist that has
been shown to inhibit vasoactive peptide-induced enhanced protein synthesis in
VSMCs, could attenuate VSMC hypertrophy in rat models of cardiac hypertrophy and to
explore the underlying mechanisms contributing to this inhibition. For these studies,
aortic VSMCs from 16-week-old SHR were used. The protein synthesis, a marker of
hypertrophy, was determined by (3H)leucine incorporation and the expression of proteins
was determined by Western blotting. Cell volume was determined by three-dimensional
confocal imaging. The protein synthesis was significantly enhanced in VSMC from SHR
as compared to WKY and C-ANP4-23 treatment attenuated the enhanced protein
synthesis to WKY control levels. In addition, the enhanced expression of the AT1
receptor as well as Gqα and PLCβ1 proteins, enhanced levels of superoxide anion (O2
-),
nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, as well as the
increased expressions of NADPH oxidase 4 (Nox4) and p47phox exhibited by VSMC from
SHR were all attenuated by C-ANP4-23 treatment. Furthermore, C-ANP4-23 also
attenuated the enhanced activation of epidermal growth factor receptor (EGF-R),
platelet-derived growth factor receptor (PDGF-R), insulin-like growth factor 1 receptor
(IGF-1R) and the enhanced phosphorylation of extracellular signal-regulated kinases 1/2
(ERK1/2), AKT and c-Src. These results indicate that C-ANP4-23, via the activation of
NPR-C, attenuates VSMC hypertrophy through its ability to decrease the overexpression
of the AT1 receptor and Gqα/PLCβ1 proteins, the enhanced oxidative stress, the
increased activation of growth factors and the enhanced phosphorylation of the
MAPK/AKT signalling pathway. Thus, it can be suggested that C-ANP4-23, an activator of
NPR-C, may be used as a therapeutic agent for the treatment of vascular complications
associated with hypertension and atherosclerosis.
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Régulation de la prolifération des cellules musculaires lisses vasculaires par l’activation in vivo du récepteur natriurétique de type CRahali, Sofiane 09 1900 (has links)
No description available.
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Modélisation quantochimiques des forces de dispersion de London par la méthode des phases aléatoires (RPA) : développements méthodologiques / Quantum chemical studies of London dispersion forces by the random phase approximation (RPA) : methodological developments.Mussard, Bastien 13 December 2013 (has links)
Dans cette thèse sont montrés des développements de l'approximation de la phase aléatoire (RPA) dans le contexte de théories à séparation de portée. On présente des travaux sur le formalisme de la RPA en général, et en particulier sur le formalisme "matrice diélectrique" qui est exploré de manière systématique. On montre un résumé d'un travail sur les équations RPA dans le contexte d'orbitales localisées, notamment des développements des orbitales virtuelles localisées que sont les "orbitales oscillantes projetées" (POO). Un programme a été écrit pour calculer des fonctions telles que le trou de d'échange, la fonction de réponse, etc... sur des grilles de l'espace réel (grilles parallélépipédiques ou de type "DFT"). On montre certaines de ces visualisations. Dans l'espace réel, on expose une adaptation de l'approximation du dénominateur effectif (EED), développée originellement dans l'espace réciproque en physique du solide. Également, les gradients analytiques des énergies de corrélation RPA dans le contexte de la séparation de portée sont dérivés. Le formalisme développé ici à l'aide d'un lagrangien permet une dérivation tout-en-un des termes courte- et longue-portée qui émergent dans les expressions du gradient, et qui montrent un parallèle intéressant. Des applications sont montrées, telles que des optimisations de géométries aux niveaux RSH-dRPA-I et RSH-SOSEX d'un ensemble de 16 petites molécules, ou encore le calcul et la visualisation des densités corrélées au niveau RSH-dRPA-I / In this thesis are shown developments in the random phase approximation (RPA) in the context of range-separated theories. We present advances in the formalism of the RPA in general, and particularly in the "dielectric matrix" formulation of RPA, which is explored in details. We show a summary of a work on the RPA equations with localized orbitals, especially developments of the virtual localized orbitals that are the "projected oscillatory orbitals" (POO). A program has been written to calculate functions such as the exchange hole, the response function, etc... on real space grid (parallelepipedic or of the "DFT" type) ; some of those visualizations are shown here. In the real space, we offer an adaptation of the effective energy denominator approximation (EED), originally developed in the reciprocal space in solid physics. The analytical gradients of the RPA correlation energies in the context of range separation has been derived. The formalism developed here with a Lagrangian allows an all-in-one derivation of the short- and long-range terms that emerge in the expressions of the gradient. These terms show interesting parallels. Geometry optimizations at the RSH-dRPA-I and RSH-SOSEX levels on a set of 16 molecules are shown, as well as calculations and visualizations of correlated densities at the RSH-dRPA-I level
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