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1
Patel, Premal Harshad. "Evolution of DNA polymerase active site /". Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/6361.
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Marcello, Matthew R. "Analysis of recombinant human prostasin carrying a serine active site mutation". Honors in the Major Thesis, University of Central Florida, 2003. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/325.
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This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucf.edu/Systems/DigitalInitiatives/DigitalCollections/InternetDistributionConsentAgreementForm.pdf You may also contact the project coordinator, Kerri Bottorff, at kerri.bottorff@ucf.edu for more information.Bachelors
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3
Wenwieser, Sandra Verena Corinna Tina. "Subunit interactions in regulation and catalysis of site-specific recombination". Thesis, University of Glasgow, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343974.
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Chitpinityol, Supannee. "Heterologous expression and site-directed mutagenesis of the enzyme chymosin". Thesis, University of Reading, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320101.
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5
Sheikh, Qaiser Iftikhar. "Exploring the structure and function of bacterial cytosine specific DNA methyltransferases using site-directed mutagenesis". Thesis, University of Sheffield, 2001. http://etheses.whiterose.ac.uk/10258/.
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Point mutations were engineered into the sequence of the multispecific DNA methyltransferase (Mtase) M. SPRI in motif IX, in order to mimic the corresponding motif IX of mono-specific Mtase. A similar approach was adopted to modify the sequence of the monospecific enzyme M. HhaI in motifs IX and X based on the available structure and as a consequence the enzyme regained methylation potential. It was thought that these changes might be sufficient to enable functional exchange of the target recognition domains (TRDs) between a mono- and a multispecific enzyme. However, insertion of various segments of TRD region from M. SPRI into the M. HhaI was not successful (Chapter 4). To establish whether mono- and multispecific Mtases are incompatible in terms of sequence exchanges, a systematic "swapping" of motifs was carried out (Chapter 5). These experiments suggested that there are some enzyme-specific structural interactions between different subunits within each class of Mtases. In second half of this thesis a bacterial two-hybrid system based on the reversible assembly of an engineered form of M. SPRI was developed (Chapter 6). However the Mtase protein does not assemble into an active species until a DNA segment encoding a leucine zipper motif is fused to each of the two halves. Co-transformation of E. coli with the plasmids expressing the C-terminal and N-terminal domains respectively resulted in the abolition of colonies on double antibiotic plates, when an mcr strain was used as host. High performance liquid chromatography was used to estimate the extent of modification of plasmids indirectly. The extent of methylation at specific sequences within a plasmid molecule was readily detected by the corresponding differential susceptibility to digestion by specific restriction enzymes. Using this approach it proved possible to detect different levels of activity produced by wild type and mutant recombinant DNA Methyltransferases with sensitivity and in a semi quantitative manner. In order to analyse the biochemical properties of Mtase, I have developed an in vitro translation-modification assay. Binary studies with the mutants (from Chapter 3 and 5) showed that there were no detectable sequence-specific recognition differences between these enzymes. Taken together, these results suggest that motif IX plays a role in general stabilisation of the enzyme core structure and has a less significant role in DNA recognition.
6
Dinda, Stephen B. "Predicting RNA Mutation Using 3D Structure". Bowling Green State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1321280932.
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Tinteroff, Gil Vanessa. "De Paracas à Nasca sur la côte du sud du Pérou : archéologie d'une mutation culturelle". Paris 4, 2008. http://www.theses.fr/2008PA040039.
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Sur la côte sud du Pérou, à l'issu du déclin de la civilisation Chavín, la culture Paracas laisse peu à peu place à la culture Nasca. Datée entre 200 avant J. -C. Et 100 de notre ère, cette période, communément appelée "transition Paracas – Nasca" sur la côte sud, est celle de nombreux changements culturels. À travers l’étude de divers contextes archéologiques de cette région, et en particulier de Necrópolis dans la péninsule de Paracas et de Cahuachi dans la vallée de Nasca, cette thèse se propose de définir les causes, les mécanismes socioculturels et les acteurs culturels des transformations qui s'opèrent sur la côte sud au cours de cette période charnière de l'histoire du Pérou préhispanique. Elle offre une définition des cultures Paracas, Topará et Nasca, de leurs origines, de leur développement et de leurs liens culturels, géographiques et chronologiquesOn the south coast of the Peru, after the decline of the Chavin civilisation, Paracas culture is slowly making way for Nasca culture. Dated between 200 BC and 100 AD, this period, commonly called "the Paracas – Nasca transition" on the south coast, is the one of numerous cultural changes. Through the study of different archaeological contexts of this region, particularly of Necrópolis in the Paracas peninsula and of Cahuachi in the Nasca valley, the purpose of this thesis is to define the causes, the sociocultural mechanisms and the cultural players of the changes occuring in the south coast during this turning point in the prehispanic Peru history. This thesis offers a definition of Paracas, Topará and Nasca cultures, of their origins, their development and their cultural, geographical and chronogical relations
8
Lefebvre, Anne. "Le site CpG dans l'ADN : impact possible des variations conformationnelles sur le taux de mutations". Châtenay-Malabry, Ecole centrale de Paris, 1996. http://www.theses.fr/1996ECAP0485.
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Nous avons analysé la structure du dinucléotide CpG en fonction de la séquence d'ADN qui le contient, par résonance magnétique nucléaire et modélisation moléculaire, pour déterminer en quoi la structure de CpG influe sur les modifications structurales induites par la méthylation de la cytosine de ce dinucléotide, et mettre en relation la structure de CpG et le taux de mutations observé sur ces sites. La permutation de ses plus proches voisins modifie fortement la conformation de CpG. Au sein de la tétrade ACGT, comme dans d(GTACGTAC)2, il adopte un grand twist, associé à une phase élevée de la guanine. Au sein de d(CATCGATG)2, la structure de CpG est beaucoup plus malléable, et il adopte des valeurs moyennes de twist et de phase de la guanine plus faibles. Mais, la conformation de CpG est également influencée par des résidus plus éloignés dans la séquence, puisque la structure qu'il adopte dans d(CTTCGAAG)2 ressemble plus à celle calculée pour d(GTACGTAC)2 que pour d(CATCGATG)2. Nous avons également montré que les effets structuraux de la méthylation de CpG dépendent nettement de la conformation initiale de ce dinucléotide : la méthylation de la cytosine centrale modifie la structure moyenne de d(CATCGATG)2 mais pas de d(CTTCGAAG)2. Enfin, dans ces deux derniers oligonucléotides, un équilibre BI/BII a été mis en évidence au niveau du squelette de CpG, la proportion de conformères BII étant plus importante dans d(CTTCGAAG)2. Remarquablement, le double mésappariement d(TpG)2 dans d(CTTTGAAG)2 adopte un équilibre BI/BII très similaire à celui observé pour CpG dans d(CTTCGAAG)2. La structure particulière de CpG dans certains contextes d'ADN, en l'absence de méthylation, suffirait donc à expliquer sa reconnaissance par des enzymes de réparation, et donc sa mutation.
9
Sada, Yoshinao. "Genetic studies on the target-site resistance to sulfonylurea herbicides in Schoenoplectus juncoides". Kyoto University, 2014. http://hdl.handle.net/2433/193553.
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Liu, Fengling. "Kinetic and Crystallographic Studies of Drug-Resistant Mutants of HIV-1 Protease: Insights into the Drug Resistance Mechanisms". Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/biology_diss/19.
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HIV-1 protease (PR) inhibitors (PIs) are important anti-HIV drugs for the treatment of AIDS and have shown great success in reducing mortality and prolonging the life of HIV-infected individuals. However, the rapid development of drug resistance is one of the major factors causing the reduced effectiveness of PIs. Consequently, various drug resistant mutants of HIV-1 PR have been extensively studied to gain insight into the mechanisms of drug resistance. In this study, the crystal structures, dimer stabilities, and kinetics data have been analyzed for wild type PR and over 10 resistant mutants including PRL24I, PRI32V, PRM46L, PRG48V, PRI50V, PRF53L, PRI54V, PRI54M, PRG73S and PRL90M. These mutations lie in varied structural regions of PR: adjacent to the active site, in the inhibitor binding site, the flap or at protein surface. The enzymatic activity and inhibition were altered in mutant PR to various degrees. Crystal structures of the mutants complexed with a substrate analog inhibitor or drugs indinavir, saquinavir and darunavir were determined at resolutions of 0.84 – 1.50 Å. Each mutant revealed distinct structural changes, which are usually located at the mutated residue, the flap and inhibitor binding sites. Moreover, darunavir was shown to bind to PR at a new site on the flap surface in PRI32V and PRM46L. The existence of this additional inhibitor binding site may explain the high effectiveness of darunavir on drug resistant mutants. Moreover, the unliganded structure PRF53L had a wider separation at the tips of the flaps than in unliganded wild type PR. The absence of flap interactions in PRF53L suggests a novel mechanism for drug resistance. Therefore, this study enhanced our understanding of the role of individual residues in the development of drug resistance and the structural basis of drug resistance mechanisms. Atomic resolution crystal structures are valuable for the design of more potent protease inhibitors to overcome the drug resistance problem.
11
Mei, Xiaonan. "HOW A SILENT MUTATION SUPPRESSES THE ACTIVITY AND IRON INCORPORATION IN SUPEROXIDE DISMUTASE". UKnowledge, 2012. http://uknowledge.uky.edu/chemistry_etds/9.
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A mutation (CTG to TTG) of FeSOD gene was found in Escherichia coli. Since they both encode leucine, it is a silent mutation. Site-‐directed mutagenesis was applied to correct the mutation, and the mutant FeSOD (before gene correction) and wild type FeSOD (after gene correction) were purified. The FeSODs from the two genes were Characterized using different assays and spectroscopic methods including EPR and CD. The requirement for the rare codon TTG may result in slowed translation and heavy demand on a scarce tRNA. Cultures expressing wild type FeSOD are better able to grow for long times after addition of IPTG and more mature to incorporate Fe atoms to the active sites than are cultures expressing the mutant gene. Moreover, the wild type FeSOD has more activity than the mutant. To our knowledge, this is the first time that a silent mutation has been demonstrated to affect metal incorporation into a metalloenzyme.
12
Lowes, Damon Anthony. "Resolution of DNA adduct formation at the nucleotide level and correlation with site specific propensity to mutation". Thesis, University of Leicester, 2002. http://hdl.handle.net/2381/30766.
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Tamoxifen, a non steroidal antioestrogen (Z-trans-1-[4-(dimethylaminoethoxy)phenyl]-1,2-diphenyl-1-butene), is widely used in the treatment of breast cancer, and is undergoing clinical evaluation as a chemopreventative in women thought to be at high risk of developing the disease. Tamoxifen is hepatocarcinogenic in rats, forming large numbers of tamoxifen DNA adducts when dosed over a period of time, but is inactive in standard genotoxicity tests. In this project I determined in vitro and in vivo DNA adduct formation at the nucleotide level from tamoxifen and selected metabolites. Sites of tamoxifen DNA adduct formation were mapped using the T4 DNA polymerase arrest and single stranded ligation assays. Following the reaction of plasmid DNA in vitro with -acetoxytamoxifen or horseradish peroxidase/H2O2 activated 4-hydroxytamoxifen, DNA adduct formation occurred predominantly on guanine. Preliminary studies in vivo also showed sites of tamoxifen DNA adduct formation on guanine with minor adduct formation on adenine. Plasmids reacted in vitro with activated 4-hydroxytamoxifen, were mutated 2 orders of magnitude more frequently than were plasmids reacted with -acetoxytamoxifen in E. coli. This occurred despite -acetoxytamoxifen forming a greater number of DNA adducts. The mutational hot spot in the bacterial lacI gene occurred in the region of the only adenine adduct for plasmids treated with activated 4-hydroxytamoxifen. These data indicated a lack of correlation between gross adduct number and mutagenic potential. This has implications for the interpretation of gross DNA adduct data.
13
Pekgoz, Gulsah. "Deletion Mutation Of Glnb And Glnk Genes In Rhodobacter Capsulatus To Enhance Biohydrogen Production". Master's thesis, METU, 2010. http://etd.lib.metu.edu.tr/upload/12612677/index.pdf.
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Rhodobacter capsulatus is a photosynthetic, purple non-sulfur (PNS) bacterium that produces biohydrogen via photofermentation. Nitrogenase enzyme is responsible for hydrogen productionduring fixation of molecular nitrogen into ammonium, hydrogen is produced. Since this process is an energetically expensive process for the cell, hydrogen production is strictly controlled at different levels. When ammonium is present in the environment, hydrogen production completely ceases. The key proteins in the regulation of nitrogenase by ammonium are two PII proteins
GlnB and GlnK. &lsquo
Hyvolution&rsquo
, 6th framework EU project, aims to achieve maximum hydrogen production by combining two hydrogen production processes
dark fermentation and photofermentation. In the first stage of the overall process, biomass is used for hydrogen production in dark fermentation process. Then, the effluent of dark fermentation is further utilized by photosynthetic bacteria to produce more hydrogen. However, the effluent of dark fermentation contains high amount of ammonium, which inhibits photofermentative hydrogen production. In order to achieve maximum hydrogen production, ammonium regulation of nitrogenase enzyme in R.capsulatus has to be released. For this purpose, all PII signal transduction proteins of R.capsulatus (GlnB and GlnK) were targeted to be inactivated by site-directed mutagenesis. The internal parts of glnB and glnK genes were deleted individually without using antibiotic cassette insertion. The successful glnB mutant was obtained at the end of mutagenesis studies. In the case of glnK mutation, the suicide vector was constructed and delivered into the cells. However, glnK mutant could not be obtained. The effect of ammonium on glnB mutant R.capsulatus was investigated and compared with wild type. Biomass of the bacterial cultures, pH of the medium and amount of produced hydrogen were periodically determined. Moreover, the concentrations of acetic, lactic, formic and propionic acids in the medium were periodically measured. Both wild type and glnB mutant grew on acetate and effectively utilized acetate. Ammonium negatively affected hydrogen production of glnB mutant and wild type. The ammonium inhibition of hydrogen production did not release in glnB mutant due to the presence of active GlnK protein in the cell
hence, inactivation of one of PII proteins was not enough to disrupt ammonium regulation of the cell. Moreover, kinetic analysis of bacterial growth and hydrogen production were done. Growth data fitted to the Logistic Model and hydrogen production data fitted to the Modified Gompertz Model.
14
O'Neill, Jason Charles Walker. "Structural studies on the B1 domain of protein L : biophysical affects of single site mutations, 3D-domain swapping, and computational redesign /". Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/4990.
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15
Scherperel, Gwynyth. "Characterization of the sequence and substrate reactivity of dihydroneopterin aldolase and its site-directed mutants by tandem mass spectrometry". Diss., Connect to online resource - MSU authorized users, 2006.
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16
Taylor, Russell Haywood. "A guanine to adenine mutation -76bp from the transcriptional start site decreases constitutive CYP1A2 expression in a novel mouse strain". Thesis, University of Ottawa (Canada), 2007. http://hdl.handle.net/10393/27924.
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Variations in the expression of drug metabolizing enzymes of the Cytochrome P450 superfamily are a principal cause of atypical reactions to therapeutics. The molecular mechanisms by which the metabolizing enzymes of the drug are regulated, and the effects of genetic variation on this regulation, are not completely understood. Cytochrome P450 1A2 (CYP1A2) is one such enzyme. The APN mouse strain has low expression of the CYP1A2 enzyme, relative to the C3H/HeJ strain. It was hypothesized that this difference in expression of the CYP1A2 was occurring as a result of a single nucleotide polymorphism at the Cyp1a2 locus. This work has demonstrated that this variation in CYP1A2 expression occurs at the level of transcription and that a single nucleotide change 76 base pairs upstream of the transcriptional start site was critical for promoter function. The mechanism of constitutive CYP1A2 expression involves a previously unidentified cis-acting element in this region.
17
Haddi, Khalid. "Studies on insecticide resistance in tuta absoluta (Meyrick), with special emphasis on characterisation of two target site mechanisms". Doctoral thesis, Università di Catania, 2012. http://hdl.handle.net/10761/1226.
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Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) is a primary pest of tomato plants and is native to South America. Since the first documented European case in 2006, it has spread throughout the Mediterranean basin and North Africa. Larval stages cause direct feeding damage and reductions to both yield and fruit quality. Chemical insecticides have been the main control tools used against T. absoluta, but decreasing efficacy has been attributed to the development of insecticide resistance.
During this study, leaf-dip bioassays were used to quantify responses of five field strains of T. absoluta to insecticides belonging to different chemical classes. The results showed significant variation in susceptibilities to organophosphates and pyrethroids, which are a major class of neurotoxic insecticides and acaricides used extensively over the last decades.
One important mechanism of resistance to pyrethroids, termed knockdown resistance (kdr), has been shown to arise through alterations (point mutations) in the para-type sodium channel protein leading to reduced sensitivity of the insect nervous system to the pyrethroids. Mutations in the Ace gene have also been reported to cause insensitivity to organophosphates.
A combination of PCR-based molecular methods and biochemical assays was used to investigate possible existence of any mutations in these two insecticides targets in several laboratory strains.
Cloning and sequencing of domains II, III, and IV of the T. absoluta sodium channel gene revealed the presence of several kdr mutations previously reported to confer reduced sensitivity in other arthropod species. These included L1014F, M918T, T929I and L925M mutations. Characterisation and sequencing of Ace gene revealed the existence of a single mutation A201S previously related to organophosphate insensitivity in several insect species.
Diagnostic tools that allow detection in individual larvae and adults were developed and used to screen field samples of diverse geographical origin and assess their distribution in global T. absoluta populations.
18
Park, Sung-Hoon. "Expression and characterization of an extremely thermostable Beta Glycosidase (Mannosidase) from the hyperthermophilic Aracheon Pyrococcus Furiosus DSM3638 and mutation studies in the active site". Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=97062.
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Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF0356) similar to the enzymes in glycoside hydrolase family 1. This beta-glycosidase, designated PFTG (Pyrococcus furiosus thermostable glycosidase), was cloned and expressed in Escherichia coli. The expressed enzyme was purified by heat treatment and Ni-NTA affinity chromatography. The gene was composed of 1452 bp encoding 483 amino acids for a protein with a predicted molecular mass of 56,326 Da. The temperature and pH optima were 100°C and 5.0 in sodium citrate buffer, respectively. The substrate specificity studies by conventional assays showed characteristics of both beta-galactosidase and beta-mannosidase activities. However, through kinetic studies by ITC (Isothermal Titration Colarimetry), this enzyme showed the highest catalytic activity in p-nitrophenyl-beta-D-mannopyranoside (pNP-Man) with kcat/Km (3.02). The enzyme showed transglycosylation and transgalactosylation activities toward cellobiose, lactose and mannooligosaccharides, that could produce GOS (galactooligosaccharides) and MOS (mannooligosaccharides) which are the important prebiotic (bifidogenic) ingredients. Sequence alignments and homology modeling of PFTG showed that the residue 150 is conserved as tryptophan in beta-glycosidase and in other related enzymes such as beta-mannosidase and beta-galactosidase. To elucidate the relationship between the substrate size and geometric shape of the catalytic site of thermophilic beta-glycosidase and category of PFTG, the Q77, the Q150 and the D206 located at the interface of the dimer were replaced with Trp and Asn. Also, to confirm the role of active sites of PFTG, the Q77/150W double mutant was created through subcloning. The mutant enzymes were successfully cloned, expressed in E. coli and showed the same temperature and pH optima at 100 °C and pH 5.0, respectively. By substituting Gln150 to Trp and Gln77/150Trp, the catalytic efficiency (kcat⁄Km) of the mutants by ITC200 on both synthetic and natural substrates were slightly changed. As compared with the wild-type enzyme, substrate specificities on the mutant enzymes were similar but with more affinity (Km) to substrates and low turnover number (kcat). To confirm the category of PFTG, the protein structural studies were performed. After the wild-type enzyme was purified, the enzyme was set up for protein crystal screening under 200 different conditions. With several hit conditions, X-ray diffraction study was performed, but during the X-ray analysis, the data was too high to obtain the reasonable data. Through protein sequence analysis of PFTG, two Lys-Lys sequences which interfere the protein crystallization were revealed. After Lys-Lys amino acids were mutated to Ala-Ala by site-directed mutagenesis, the mutant enzymes were set up and incubated for crystallization. Clear protein crystals were not obtained, but the computer 3D model structure indicated that this enzyme was similar to that of beta-glycosidase from Thermosphaera aggregans.L'analyse génomique de la Pyrococcus furiosus, hyperthermophile archaeon a révélé la présence d'un cadre de lecture ouvert (ORF PF0356) similaire aux enzymes de la famille glycoside hydrolase 1. L'enzyme beta-glycosidase, désigné PFTG (Pyrococcus furiosus glycosidase thermostable), a été cloné et exprimé dans Escherichia coli. L'enzyme exprimée a subi un traitement thermique et a été purifié par une chromatographie d'affinité au nickel. Son gène a une taille de 1452 pb et code pour une protéine de 483 acides aminés. Sa masse moléculaire prédite est de 56,326 Da. Les conditions optimales de l'activité de cette enzyme ont été définies dans un tampon citrate de sodium à pH 5,0 et à une température de 100 °C. Les études de spécificité du substrat ont démontré des caractéristiques similaires aux activités enzymatiques de la beta-galactosidase et de beta-mannosidase. À partir des résultats obtenues de cinétique par ITC (titration isotherme colarimetry), cette enzyme a révélé avoir une activité catalytique la plus élevé pour le substrat p-nitrophényl-beta-D-mannopyranoside (pNP-Man) avec un ratio Kcat/Km de 3,02. L'enzyme a montré avoir des activités de transglycosylation et de transgalactosylation envers le cellobiose, le lactose ainsi que les mannooligo-saccharides. Ce qui pourraient produire des GOS (galacto oligosaccharides) et des MOS (mannooligosaccharides) qui sont d'importants ingrédients prébiotiques (bifidogène). Les alignements de séquences et de modélisation par homologie de la PFTG ont révélé que le résidu 150, qui est le tryptophane, est conservé dans la beta-glycosidase ainsi que dans d'autres enzymes apparentés tels que la beta-galactosidase et le beta-mannosidase. Pour élucider la relation entre la taille du substrat et la forme géométrique du site catalytique de l'enzyme thermophilique beta-glycosidase ainsi que la catégorie de PFTG, les acides aminés Gln 77, Gln 150 et Asp 206 situées à l'interface du dimère ont été remplacés par mutagenèse dirigée. Ainsi, le tryptophane a été substitué aux résidus Gln 77 et 150 et l'asparagine a été substituée au résidu Asp 206. De plus, le double mutant Q77/150W a été créé par sous-clonage pour confirmer le rôle des sites actifs de PFTG. En effet, le gène PFTG de type sauvage a été muté, cloné puis exprimé dans E. coli. L'enzyme mutante exprimée à démontrer la même activité dans les mêmes conditions que l'enzyme natif, soit de tampon, de température et de pH. En substituant Gln150 et Gln77 par des tryptophanes, l'efficacité catalytique (Kcat/Km) du mutant mesuré par ITC200 a été légèrement modifié sur les substrats synthétiques et naturels. En comparant avec l'enzyme natif, la spécificité de l'enzyme mutant pour les substrats a été similaire mais avec une plus grande affinité (Km) pour les substrats et un faible Kcat. Pour confirmer la catégorie des PFTG, des études sur la structure de la protéine ont été effectuées. Après avoir purifié l'enzyme de type sauvage par chromatographie d'affinité au nickel et par chromatographie sur gel (GPC), l'enzyme purifiée a été préparée à des fins de dépistage de cristaux et mis à l'épreuve dans les 200 conditions différentes.Au cours des analyses aux rayons X, les données du facteur de diffraction se sont avérées trop élevés pour donner des résultats significatifs. Par contre, l'analyse de séquence protéique de la PFTG native a révélé deux liaisons Lys-Lys qui ont entravé la cristallisation de la protéine. Après que la liaison d'acides aminés Lys-Lys a été substituée par une liaison Ala-Ala par mutagenèse dirigée, l'enzyme mutant a été conduit à des analyses de cristallographie. Il n'a pas été possible d'obtenir des cristaux de protéine. Par contre, la modélisation par ordinateur de la structure 3D a indiqué que cette enzyme est similaire à celle de la beta-glycosidase de Thermosphaera aggregans.
19
Bouchart, Franck. "Impact des mutations OPG chez Erwinia chrysanthemi : recherche de suppresseurs et analyse protéomique des mutants". Lille 1, 2006. https://ori-nuxeo.univ-lille1.fr/nuxeo/site/esupversions/df8106c9-51b3-47fd-9d4c-9aba9e38f18e.
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Erwinia chrysanthemi est une entérobactérie phytopathogène pectinolytique faisant partie de la subdivision γ des protéobactéries. Elle infecte un large spectre de plantes hôtes engendrant la pourriture molle. L'apparition de cette pourriture molle dépend de la synthèse et de la sécrétion d'une batterie d'exoenzymes (cellulases, protéases et pectate-Iyases) capable de dégrader les composants de la paroi des cellules végétales. Néamnoins, l'expression de nombreux autres gènes est nécessaire à la virulence de cette bactérie. En effet, les mutants déficients en glucanes périplasmiques osmorégulés (OPG) d'E. Chrysanthemi sont totalement non virulents. Les OPG sont constitués de 5 à 12 résidus de glucose liés en β,1-2, branchés en β,1-6 et sont d'autant plus abondants que l'osmolarité du milieu est faible. Ces mutants présentent un phénotype pléïotrope: ils sont hypersensibles aux sels biliaires, la synthèse d'exopolysaccharides est augmentée, la synthèse et la sécrétion d"exo-enzymes ainsl que la motilité sont réduites. Un phénotype similaire, mais atténué, est observé chez les mutants du système ToI. Les OPG comme les protéines ToI apparaissent nécessaires au maintien de l'intégrité de l'enveloppe. Des mutations suppressives restaurant la motilité des mutants opg ont été isolées. Elles rétablissent l'essentiel du phénotype sauvage (virulence sur tubercules de pommes de terre, sécrétion des exo-enzymes, résistance aux sels biliaires, colonies redevenues non muqueuses et motilité, à l'exception de la virulence sur feuille d'endive). Une des mutations identifiées rcsC2 supprime également les phénotypes des mutants toICette mutation rcsC2 affecte un système capteur-régulateur rcsBCD impliqué dans la régulation de gènes cibles en réponse à un ou des signaux de nature inconnue et nécessaire au pouvoir pathogène des bactéries chez les animaux et chez les plantes. Les fonctions des gènes cibles régulés par ce système, correspondent pour une grande part aux fonctions altérées par l'absence d'OPG chez E. Chrysanthemi. Ces résultats nous laissent penser que les OPG pourraient être une des molécules captées par le système RscBCD. De plus, en parallèle à cette étude, une analyse protéomique comparative du mutant opgG par rapport à la souche sauvage d'E. Chrysanthemi, nous a permis de mettre en évidence une altération importante des fonctions cellulaires allant des structures de l'enveloppe qui était notre seule hypothèse quant à l'impact de l'absence d' OPG, mais nous constatons également une altération d'expression des protéines du métabolisme énergétique, de certains systèmes de transport et de protémes impliquées dans la dégradation et la conformation des protéines souvent induites en réponse à divers stress. L'ensemble de ces résultats nous laisse penser que les OPG pourraient être des molécules captées par un jeu de systèmes à deux composants afin de permettre à la bactérie de répondre à une multitude de stress rencontrés dans l' environnement
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Radev, Zlatko. "Site-directed nucleases as tools for genome editing in fish". Thesis, Paris 11, 2014. http://www.theses.fr/2014PA112422.
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L'application des techniques de séquençage à haut débit dans les dernières années a conduit à l'obtention de la séquence de génomes complets de plusieurs organismes. Le développement de nouveaux outils de génétique inverse était donc souhaitable afin de faire un usage optimal des données accumulées. Les nucléases hautement spécifiques représentent un outil unique pour induire des modifications ciblées du génome in vivo. L'induction d'une cassure double brin dans l'ADN est réparée soit par la voie de jonction d’extrémités nonhomologues soit par la voie très fidèle de la recombinaison homologue. Le ciblage d'un locus précis avec une nucléase spécifique stimule fortement la réparation de l'ADN, qui peut être utilisé pour induire des modifications ciblées dans le génome. Dans ma thèse, je vise à fournir une preuve de prinicipe pour l'utilisation des méganucléases et des transcription activator like effector nucléases (TALENs), deux classes communes de nucléases très spécifiques, comme nouveaux outils d'édition du génome chez le medaka, Oryzias latipes, et le poisson zèbre, Danio rerio. J'ai d’abord trouvé les conditions optimales d'utilisation de ces nucléases dans nos modèles de poissons. J'ai à cette occasion également développé une méthode très sensible et rapide pour la détection de modifications génomiques ciblées. J'ai ensuite induit des mutations au sein de trois gènes différents chez le poisson zèbre avec des TALENs. Les mutations dans le gène col6a1 ont conduit à la mise en évidence pour la première fois d’une technique de modification d’un site d’épissage d’un gène de poisson zèbre à l’aide d’une nucléase ciblée. Ce travail nous a permis d’établir une lignée de poisson avec une mutation dans le collagène VI alpha 1 qui est similaire à une mutation fréquemment trouvée chez les patients humains atteints de myopathie de Bethlem. De même, j’ai pu induire des mutations dans le gène nle1 du poisson zèbre qui vont permettre la mise en place de lignées de poissons mutants de ce gène. En outre, j'ai pu montrer qu’un nouveau type de nucléase, une TALEN Compact, était actif sur une cible chromosomique chez le poisson zèbre. En conclusion, les études que j'ai effectuées ont apporté la preuve de principe pour l'activité de TALENs et Compact TALENs ainsi que la première démonstration de modification de l'épissage à l’aide d’une TALEN chez le poisson zèbre et ont abouti à la mise en place d'une lignée de poisson dont le phénotype est proche d’un syndrome humain ouvrant la voie à la création de modèles pour d’autres mutations de ce type. Pour finir, je discute dans cette thèse des conditions permettant un usage le plus efficace des nucléases ciblées pour la génération de mutants et l’édition du génomeThe application of high throughput sequencing techniques in the recent years has led to obtaining the full genome sequences of many organisms. The development of novel tools for reverse genetics was thus desirable to make optimal use of the accumulated data. Site directed nucleases represent a unique platform to induce targeted genome modifications in vivo. Targeting a precise locus with a highly specific nuclease stimulates DNA repair, which can be harnessed for genome editing. Induction of a double strand break in DNA is repaired by either the error prone pathway of nonhomologous end joining or the high fidelity pathway of homologous recombination in the cell. Both mechanisms can be used to insert foreign DNA into the genome of the host. In my thesis, I aimed to provide proof of principle for the use of meganucleases and transcription activator like effector nucleases (TALENs), two common classes of site directed nucleases, as novel tools for genome editing in medaka, Oryzias latipes, and zebrafish, Danio rerio. During the first years of my thesis, I found the optimal conditions to use these nucleases in our fish models. I also developed a very sensitive and rapid method for detection of targeted genome modifications. I then induced mutations at three different endogenous loci in zebrafish with TALENs. The mutations in the col6a1 gene led to the first demonstration of splicing site modification in zebrafish using a TALE nuclease. This allowed the establishment of a fish line with a mutation in type VI collagen alpha 1 chain homologous to one mutation frequently found in human patients with Bethlem myopathy. Then I generated mutations in the nle1 gene which are heritable and from which establishment of mutant fish lines is in progress. In addition, by using the method for detection of targeted genome modifications I developed, I showed that a novel type of nuclease, a Compact TALEN, was active on a chromosomal target in zebrafish. In conclusion, the studies I performed provided proof of principle for the activity of TALENs and Compact TALENs as well as the first demonstration of TALEN-Mediated modification of splicing in zebrafish and resulted in the establishment of a fish line with mutated collagen VI. Induction of heritable mutations in the nle1 gene in zebrafish was also confirmed. Additionally, I proved that the choice of expression vector is crucial for the synthesis of active site directed nucleases for use in fish and established a novel efficient method for detection of targeted genomic mutations
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Borgato, Ednaldo Alexandre. "Identificação de Amaranthus palmeri, caracterização da resistência múltipla a herbicidas inibidores da ALS e da EPSPS e controle químico baseado no uso das novas tecnologias transgênicas". Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/11/11136/tde-16052018-125757/.
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A planta daninha Amaranthus palmeri é nativa dos Estados Unidos, porém foi pela primeira vez relatada no Brasil no ano de 2015. Embora comprovadamente com resistência múltipla aos herbicidas inibidores da ALS e da EPSPS, até o momento não foram investigadas as bases moleculares da resistência. Além disso, por causa da recente introdução da planta daninha no país, alternativas de manejo com culturas tolerantes a herbicidas necessitam ser estudadas. Sendo assim, os objetivos desse trabalho são de caracterizar a espécie de planta daninha introduzida no país, identificar os mecanismos de resistência aos herbicidas inibidores da ALS e da EPSPS presentes no biótipo, e propor abordagens de manejo em ambientes dos novos eventos transgênicos resistentes a herbicidas. Um bioensaio utilizando marcadores genéticos foi desenvolvido para confirmar que a população coletada no estado do Mato Grosso (BR-R) é A. palmeri, e não A. tuberculatus, outra espécie dióica do gênero Amaranthus. Os resultados de experimentos de curvas de dose-resposta e acúmulo de chiquimato indicaram que a BR-R possui alto nível de resistência, com DL50 de 4.426 e 3.400 g glyphosate ha-1 no primeiro e segundo experimento, respectivamente, mais que o dobro da dose típicamente recomendada para o controle da espécie e, adicionalmente, observou se acúmulo mínimo de chiquimato a concentração de 1 mM nos tecidos das plantas tratadas com o herbicida. BR-R também foi resistente a herbicidas dos grupos químicos das sulfoniluréias e imidazolinonas. O mecanismo de resistência ao glyphosate encontrado nesta população foi a super expressão gência, através do aumento no número de cópias do gene da EPSPS no genoma da planta BR-R, entre 50 e 179 cópias adicionais. Além disso, duas substituições de aminoácidos foram observadas na sequência da ALS, W574L e S653N, conferindo resistência tanto a sulfoniluréias quanto a imidazolinonas. No experimento utilizandos os herbicidas correspondentes às culturas geneticamente modificadas com novos traits de tolerância a herbicidas observou se, de uma forma geral, que as associações de herbicidas apresentaram níveis de controle mais satisfatórios. Assim, esta pesquisa confirma a introdução de da espécie A. palmeri no Brasil, assim como a resistência múltipla aos herbicidas inibidores da EPSPS e da ALS. Seu manejo é mais eficaz através da associação de herbicidas, garantindo assim o uso racional das novas tecnologias de culturas geneticamente modificadas com tolerância a herbicidas.Palmer Amaranth (Amaranthus palmeri) is a weed species native to the United States, but it was reported in Brazil for the first time in 2015. Despite this population being resistant to EPSPS and ALS inhibitors, the molecular basis of its multiple resistance is unknown up to date. Because of this species introduction to Brazil, alternatives of management with the new herbicide-tolerant crops technologies need to be studied. The objectives of this research are to characterize the weed species introduced to Brazil, identify the mechanisms conferring resistance to ALS and EPSPS inhibitors herbicides, and to propose management approaches in environments with the new genetically modified herbicide-tolerant crops. A genotyping bioassay using genetic markers was developed to confirm that the species collected in the state of Mato Grosso (BR-R) is indeed A. palmeri and not A. tuberculatus, another dioceous species in the Amaranthus genus. Dose-response experiments and shikimate accumulation bioassay data indicate high level of resistance, with LD50 of 4,426 and 3,400 g glyphosate ha-1 in the first and second experiments, respectively, higher than the double rate tipically recommended to control it, and minimal accumulation in BR-R with 1 mM of glyphosate in treated plants in the leaf disks assay. BR-R also was resistanto to sulfonilurea and imidazolinone herbicides. The mechanism conferring resistance to glyphosate identified in this population was gene amplification, with increased EPSPS copy number - between 50 and 179 more copies in BR-R. Besides, two target-site mutations were identified in the ALS gene sequencing, W574L and S653N, conferring resistance to sulfonilureas and imidazolinones. The weed control experiment, overal, herbicide tank mixtures achieved higher levels of control. Therefore, this research confirms the introduction of A. palmeri to Brazil, as well as its multiple resistance to EPSPS and ALS inhibitor herbicides. Its control is more efficient with herbicide mixtures, which guarantees more susteinable use of the new herbicide-tolerant crop technologies.
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Simões, Sarah Capelupe. "Caracterização bioquímica e farmacológica de receptores AT1 de angiotensina II contendo mutações relacionadas à fibrilação atrial em humanos". Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/17/17131/tde-01022016-152915/.
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Os receptores acoplados à proteína G (GPCRs) são proteínas integrais de membrana caracterizados por possuírem sete alfa-hélices transmembranares. Esses receptores são importantes alvos de estudos biomédicos e aproximadamente 40% dos medicamentos atualmente comercializados agem sobre estes receptores. O receptor de Angiotensina II do tipo 1 (AT1) é um GPCR e o principal mediador do Sistema Renina-Angiotensina que tem como principal efetor o octopeptídeo Angiotensina II (AngII). Recentemente foi descrito que as mutações A244S e I103T-A244S no receptor AT1 podem estar relacionadas com a predisposição à fibrilação atrial. Neste trabalho foi realizada a construção, caracterização bioquímica e farmacológica destes mutantes, bem como do mutante I103T, com o objetivo de compreender como a funcionalidade desses receptores mutantes poderiam contribuir para a predisposição à fibrilação atrial. Os mutantes I103T e I103T-A244S revelaram ser mais eficientes e potentes que o receptor selvagem em aumentar os níveis de cálcio intracelular em resposta à AngII. Todos os mutantes estudados apresentaram baixa eficiência quanto à ativação da via das MAPKs e apresentaram comportamento diferente do receptor selvagem quando bloqueados com o antagonista Losartan, seletivo para o receptor AT1 e muito usado na clínica como medicamento anti-hipertensivo. Esses dados ressaltam a relevância do estudo tanto em termos de melhor compreender as bases moleculares da relação entre as mutações e a doença, bem como possível prevenção ao uso de medicamentos que possam interagir e agir diferentemente em receptores com essas mutações.G-protein coupled receptors (GPCRs) are integral membrane proteins characterized by having seven transmembrane alpha-helices. These receptors are important targets of biomedical studies and approximately 40% of currently marketed drugs act on such receptors. The angiotensin II type 1 receptor (AT1) is a GPCR and the main mediator of the Renin-Angiotensin System whose main effector is the octapeptide Angiotensin II (Ang II). It was recently described that I103T and A244S mutations in the AT1 receptor may be related to the susceptibility to atrial fibrillation. In this study we carried out the construction of these mutants and their biochemical and functional characterization. The I103T and I103T/A244S mutants were shown to be more efficient and potent than the wild-type receptor on the increase of intracellular calcium levels. All mutants showed lower efficcacy for MAPK pathway activation and showed different behavior when compared to the wild-type receptor after antagonism with Losartan. These data highlight the relevance of the present study concerning a better understanding of the molecular basis of cardiovascular diseases and showing that conventional therapies for certain diseases may lead to adverse effects on patients carrying point mutations on the receptor sequence.
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Smith, Adam N. "Reduced Chemical Weed Control Options in Virginia for Corn and Turfgrass and Characterization of Sorghum halepense Expressing Multiple Resistance to Nicosulfuron and Glyphosate". Diss., Virginia Tech, 2014. http://hdl.handle.net/10919/56957.
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Sustainable weed control in managed agricultural systems requires the judicious use of multiple weed control tactics and prevents over-reliance on any one tactic. In this context, sustainable weed management plays a critical role in the mitigation of one of agriculture's most pressing problems- herbicide resistance. Research conducted in Virginia sought to explore the effects of integrating multiple weed management tactics in corn and cool-season turfgrass. Additionally, research was conducted to confirm nicosulfuron and glyphosate herbicide resistance in Virginia johnsongrass and elucidate the molecular mechanisms conferring those resistances. Rye and hairy vetch cover crop residues, combined with reduced rates of preemergence herbicide and postemergence glyphosate applications, were shown to provide sufficient weed control and corn yield. Cover crop type or residue level did not augment weed control in corn production systems, but the use of glyphosate was essential for late-season weed control. Rye and vetch biculture as a cover crop increased corn yield compared to rye cover crop alone. In cool-season turfgrass, the addition of reduced preemergence herbicide rates to corn gluten meal, an organic herbicide product, reduced crabgrass 25%. Moreover, control was dependent on herbicide choice. Herbicides applied at half of recommended labeled rates or less did not control crabgrass at a commercially-acceptable level, regardless of corn gluten meal addition. In field experiments, Virginia johnsongrass expressed resistance to nicosulfuron and glyphosate. Glyphosate at 0.88 kg ae ha-1 controlled johnsongrass 65%. Nicosulfuron at 0.14 kg ai ha-1 controlled the same population 10%. Greenhouse experiments confirmed differential sensitivity of putative herbicide-resistant johnsongrass seedlings to nicosulfuron and glyphosate when compared to a susceptible population. Herbicide resistance was not conferred via target-site mutation. Five ALS-gene site mutations were confirmed absent in Virginia johnsongrass, while three others were located in coding regions that could not be elucidated in johnsongrass. Further investigations showed glyphosate resistance was not conferred via reduction in herbicide absorption or translocation. The susceptible johnsongrass caused an increase in a polar metabolite at Rf = 0.17 with concomitant reduction in glyphosate over time. Although the mechanism is not clear, these data suggests that glyphosate resistance in johnsongrass may be associated with differential metabolism.Ph. D.
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Mazza, Catherine. "17-hydroxystéroïde déshydrogénase humaine de type I : analyse des relations structure-fonction par mutagenèse dirigée et cristallographie des rayons X". Université Joseph Fourier (Grenoble ; 1971-2015), 1997. http://www.theses.fr/1997GRE10214.
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La 17-hydroxysteroide deshydrogenase de type 1 (17-hsd1) est responsable de la synthese de l'stradiol, strogene biologiquement actif implique dans le declenchement et la proliferation des cancers du sein hormono-dependants. Cette enzyme appartient a la famille des deshydrogenases reductases a chaine courte dans laquelle les residus ser142 tyr155 et lys159, tres conserves, sont supposes intervenir dans le transfert de proton. La 17-hsd1 presente une specificite exclusive, mais inexpliquee vis-a-vis des steroides possedant un cycle a aromatique. De plus, elle utilise, in vitro, aussi bien le nad#+ que le nadp#+ comme cofacteur. Au cours de ce travail, nous avons tente d'identifier les interactions qui regissent ces specificites et de proposer un mecanisme reactionnel. Pour ce faire, des mutants s142a, y155f, s142a/y155f, k159m, h221l, h221q et -17-hsd1 (deletion des 38 residus c-terminaux) ont ete construits et exprimes dans le systeme baculovirus-cellules d'insecte. Les parametres de cinetique enzymatique de ces mutants ont ete compares a ceux de l'enzyme sauvage et une etude structurale a ete menee sur certains de ces mutants. Ainsi, des complexes h221l/nadp#+/stradiol, -17-hsd1/nadp#+/estradiol, h221l/nad#+ et h221q/stradiol ont ete resolus respectivement a 2. 7 a, 1. 9 a, 3. 0 a et 2. 7 a de resolution. Ces structures nous montrent une image detaillee des interactions enzymes-nadp#+ dans lesquelles intervient une boucle mobile (191-199), desordonnee dans toutes les structures precedemment decrites. Stabilisee uniquement en presence de cofacteur, cette boucle se rabat sur le site actif et le protege du solvant. De plus, le residu phe192 stabilise le groupement nicotinamide par un contact hydrophobe fort. Conjointement, ces deux elements favorisent le transfert d'hydrure. Par ailleurs, le groupement 2-phosphate du nadp#+ est stabilise par deux interactions ioniques avec les residus arg37 et lys195 et par une liaison hydrogene avec la ser11. Cette stabilisation demontre la preference de l'enzyme pour le coenzyme nadp(h) et souligne l'originalite de la 17-hsd1 dans laquelle un des deux residus basiques est situe en dehors du motif rossmann fold. De plus, l'analyse des proprietes catalytiques et du mode de liaison de l'stradiol pour les deux mutants h221l et h221q suggere que la liaison hydrogene normalement observee entre le residu his221 et l'oxygene o#3 du steroide joue un role primordial dans le determinisme de la specificite de l'enzymze vis-a-vis des substrats possedant un cycle a aromatique. Enfin, l'analyse structurale du site catalytique des complexes ternaires associee a la perte d'activite enzymatique des mutants s142a, y155f, s142a/y155f et k159m nous a permis de proposer un mecanisme catalytique dans lequel le residu tyrosine jouerait le role d'acide en donnant son proton a l'oxygene o#1#7, le residu serine stabiliserait l'intermediaire reactionnel et contriburait a reduire le pka de la tyrosine alors que le role principal de la lysine serait de stabiliser le groupement nicotinamide.
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Hegazy, Usama M. "Structure-Function Relationships of Pi Class Glutathione Transferase Studied by Protein Engineering". Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7146.
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Galindo, ramirez Martha Liliana. "Un monde en mutation : jeunesse, internet et politique : les cas du mouvement étudiant MANE en Colombie et du mouvement Acampa Sampa Ocupa Sampa au Brésil : (2011)". Thesis, Université Grenoble Alpes (ComUE), 2016. http://www.theses.fr/2016GREAH033.
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Cette thèse analyse les transformations des pratiques politiques des jeunes liées à l’usage d’Internet, en particulier de Facebook, en 2011 dans le cas du mouvement pour l’éducation Mane -Mesa Amplia Nacional Estudiantil- en Colombie et du mouvement d’occupation Acampa Sampa Ocupa Sampa au Brésil. Ce travail intègre des entretiens, l’élaboration de bases de données issues des pages Facebook et l’étude des dynamiques en ligne et hors ligne en soulevant des singularités et des enjeux méthodologiques. Il examine des modalités d’appropriation de Facebook ainsi que le statut de la jeunesse, les rapports au politique et la place d’internet. Les entretiens en face-à-face et l’analyse des bases de données des pages Facebook ont permis d’établir les contenus des revendications, les appels des mouvements, la mise en évidence occasionnelle des mécontentements et des disputes, l’administration singulière et l’utilisation différenciée des divers outils numériques, les usages des réseaux sociaux qui ont eu une grande capacité de rassembler et qui ont aussi été au cœur des polémiques autour des intérêts personnels, partisans ou collectifs des mouvements. Les mouvements étudiés partagent des caractéristiques semblables tout en ayant des singularités par rapport à : leur mode d’émergence, leurs liens avec des mobilisations précédentes, leurs principes, leurs revendications, leurs modes d’organisation et d’appropriation d’Internet et du réseau Facebook et leurs relations à la rue et au web, à l’espace et au temps, à la visibilité et l’invisibilité. La question de « l’apartisanisme » est présente dès l’origine, que ce soit à travers l’activité des mouvements hors ligne ou en ligne. Elle est en partie la cause de conflits qui ont eu lieu à l’intérieur des mouvements. L’affirmation de « l’apartisanisme » a participé au succès des mouvements et elle explique également leur essoufflement. L’analyse de likes, de partages et de commentaires a permis de repérer : l’enthousiasme qui se manifeste au moment de leur essor, les moments de déclin, les rapports avec la police et les autorités locales, les échanges et débats internes, les appels « officiels » des administrateurs des pages et les réponses de sympathisants ou des personnes critiques à l’égard de certaines manières de décider et d’orienter les mouvements. L’approche de la jeunesse, en tant que catégorie construite et déterminée par son contexte, est bouleversée d’une part, par l’irruption du numérique et, d’autre part, par la remise en cause du moratoire social. En faisant le lien entre ces deux bouleversements, un troisième élément apparaît conduisant à un nouveau questionnement. La condition de jeunesse semble s’élargir (plus de temps et de conditions matérielles pour les loisirs et l’oisiveté) alors qu’elle se rétrécie avec les orientations économiques actuelles qui amplifient l’importance du marché et démantèlent peu à peu les politiques favorables au moratoire social. Dans ce contexte où l’accentuation des contraintes est allée de pair avec l’émergence de nouvelles possibilités d’action, ces mouvements sociaux ont eu comme objectif de dénoncer le fonctionnement restreint de la démocratie, de remettre en cause la concentration de la richesse et du pouvoir, d’expérimenter de nouvelles formes de protestation et de mobilisation en mettant en rapport les problèmes locaux avec des dynamiques internationales, en participant à une mouvance transnationale de contestation. Ce travail insiste sur la nécessité de surmonter l’opposition réel versus virtuel, d’éviter de traiter le numérique comme le miroir de l’univers non numérique ou de réduire chacune de ces dimensions l’une à l’autre. Il souligne la portée, les spécificités et les imbrications des ordres de réalité en ligne et hors ligne et constate une cohabitation entre des formes d’action différentes, avec à la fois des transformations et des continuitésThis thesis analyzes the transformations of political practices of youth related to Internet use, especially Facebook, in 2011 in the cases of the student movement Mane -Mesa Amplia Nacional Estudiantil- in Colombia and the Occupy movement Acampa Sampa Ocupa Sampa in Brazil. This work incorporates interviews, development of databases from Facebook pages and the study of the dynamics online and offline and discuss the singular methodological aspects. This study examines the appropriation modalities of Facebook as well as the status of youth, politics and internet.By face-to-face interviews and an analysis of databases from the Facebook pages, this research establishes the content of the claims, calls of the movements, the occasional disagreements and disputes, the singular administration and utilization of various digital tools, the uses of social networks who had a great capacity to gather and also to be at the heart of the controversy around personal, collective or partisan interests inside the movements.Studied movements share similar characteristics and reveal singularities relative to: their mode of emergence, their links with previous protests, their principles, their demands, their modes of organization and appropriation of the Internet and the Facebook network and their relationship to the street and the web, the space and the time, the visibility and the invisibility.The issue of "apartisanisme" is present from the beginning through the offline and online activity of the movements. It is partly the cause of conflicts that took place inside them. The affirmation of "the apartisanisme" participated in the success of the movements and it also explains their stagnation.Analysis of likes, shares and comments allowed to identify: the enthusiasm manifested at the rise period, the moments of decline, relations with the police and local authorities, exchanges and internal debates, 'official' calls from administrators of pages and responses to support and to criticize some ways to decide and guide movements.The approach on youth, as a category built and determined by its context, is altered, on the one hand, by the irruption of the digital and, on the other hand, by the questioning of the social moratorium. By making the link between these two changes, a third element emerges and suggests new questions. The condition of youth seems to expand (more time and material conditions for leisure) while it is reduced according to the current economic guidelines which amplify the importance of the market society and dismantle little by little the moratorium social policies.In this context where emphasis constraints and emergence of new possibilities for action go together these social movements’ objective was to denounce the democracy operation restricted, the concentration of wealth and power, to experiment new forms of protest and mobilization linking local problems with international dynamics by participating in a transnational protest movement.This work points out the need to surpass the opposition real versus virtual, to avoid treating digital world as the mirror of the non-digital world or reduce each of these dimensions to the other. It highlights the scope, the specificities, and the interweaving of the online and offline reality orders and it establishes coexistence between different forms of action, including the continuities and transformations
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Cavallin, Mara. "Physiopathologie moléculaire et cellulaire des anomalies du développement du cortex cérébral : le syndrome d'Aicardi WDR81 mutations cause extreme microcephaly and impair mitotic progression in human fibroblasts and Drosophila neural stem cells TLE1, a key player in neurogenesis, a new candidate gene for autosomal recessive postnatal microcephaly Mutations in TBR1 gene leads to cortical malformations and intellectual disability Aicardi syndrome: Exome, genome and RNA-sequencing of a large cohort of 19 patients failed to detect the genetic cause Recurrent RTTN mutation leading to severe microcephaly, polymicrogyria and growth restriction Recurrent KIF2A mutations are responsible for classic lissencephaly Recurrent KIF5C mutation leading to frontal pachygyria without microcephaly Rare ACTG1 variants in fetal microlissencephaly De novo TUBB2B mutation causes fetal akinesia deformation sequence with microlissencephaly: An unusual presentation of tubulinopathy A novel recurrent LIS1 splice site mutation in classic lissencephaly Further refinement of COL4A1 and COL4A2 related cortical malformations Prenatal and postnatal presentations of corpus callosum agenesis with polymicrogyria caused By EGP5 mutation Delineating FOXG1 syndrome from congenital microcephaly to hyperkinetic encephalopathy Delineating FOXG1 syndrome: From congenital microcephaly to hyperkinetic encephalopathy". Thesis, Sorbonne Paris Cité, 2019. https://wo.app.u-paris.fr/cgi-bin/WebObjects/TheseWeb.woa/wa/show?t=2213&f=18201.
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Les malformations du cortex cérébral (MDC) représentent une cause importante de handicap et d'épilepsie pharmaco-résistante. Le séquençage à haut débit a permis une amélioration considérable de l'identification des bases moléculaires des MDC non syndromiques. Toutefois, certaines formes, notamment les MDC complexes, demeurent inexpliquées. Mon projet de thèse a pour objectif de progresser dans la compréhension des MDC complexes en utilisant deux modèles : les microlissencéphalies (MLIS) et le syndrome d'Aicardi (AIC), une forme syndromique particulière associant des malformations de l'oeil et du cerveau uniquement rapporté chez les filles. L'étude par séquençage d'exome en trios de 16 familles MLIS m'a permis d'identifier et de caractériser un nouveau gène, WDR81, impliqué dans le cycle cellulaire. Par la même stratégie, j'ai pu identifier un variant homozygote pathogène dans TLE1, un partenaire majeur de FOXG1 dans la balance prolifération/différenciation de progéniteurs neuronaux, dans une famille consanguine de microcéphalie postnatale dont le phénotype est proche du syndrome FOXG1. En parallèle, mes travaux ont permis de préciser les spectres phénotypiques associés à RTTN, EPG5, COL4A1, COL4A2, TBR1, KIF5C, KIF2A et FOXG1. La deuxième partie de mon projet avait pour objet l'identification des bases moléculaires du syndrome d'Aicardi à partir d'une cohorte internationale de 19 patientes. Après avoir exclu un biais d'inactivation du chromosome X et la présence de microremaniements chromosomiques, j'ai réalisé un séquençage d'exome en trio. Aucun variant récurrent n'a été retrouvé dans les séquences codantes. Dans un second temps, j'ai testé une approche combinant les données du séquençage de génome et l'analyse du transcriptome (RNA-Seq) sur fibroblastes, me permettant d'identifier des transcrits dérégulés qui étaient impliqués dans le développement du cerveau et de l'oeil. J'ai comparé les résultats de cette analyse avec ceux de l'analyse du génome dans le but d'identifier des variants dans ces gènes candidats. En conclusion, mon travail de thèse a permis d'améliorer la connaissance des bases moléculaires des MDC complexes et d'ouvrir des perspectives de nouveaux mécanismes tels que ceux engageant les gènes WDR81 et EPG5, et le rôle des endosomes et de l'autophagie dans les MDC, et aussi TLE1 comme nouvelle cause de microcéphalies postnatales. Mes travaux ont également permis de générer une collection de données de séquençage haut débit (WES, WGS et RNA-Seq) qui seront mises en commun dans le cadre d'un consortium international afin de développer des nouvelles stratégies d'analyse en particulier pour les séquences non codantes. Cette approche permettra également d'ouvrir la voie vers la compréhension des mécanismes cellulaires impliqués dans la formation du cerveau et de l' œilMalformations of cortical development (MCD) are a major cause of intellectual disability and drug-resistant epilepsy. Next Generation Sequencing (NGS) has considerably improved the identification of the molecular basis of non-syndromic MCD. However, certain forms, including complex MCD, remain unexplained. My PhD project aimed to improve the understanding of complex MCD using two disorders: Microlissencephaly (MLIS) and Aicardi Syndrome (AIC), the latter associating brain and eye malformations and only reported in girls. Trio Whole Exome Sequencing (WES) performed in 16 MLIS families allowed me to identify and functionally characterize a new MLIS gene, WDR81, in which mutations lead to cell cycle alteration. Moreover, using the same strategy, I was able to identify a pathogenic homozygous variant in TLE1 in a patient from consanguineous family with a postnatal microcephaly, suggestive of a FOXG1-like presentation. Interestingly, TLE1 is a major partner of FOXG1, a gene involved in maintaining the balance between progenitor proliferation and differentiation. In parallel, my work allowed me to redefine the phenotypic spectrum associated with RTTN, EPG5, COL4A1 and COL4A2, TBR1, KIF5C, KIF2A and FOXG1. The second part of my PhD program was aimed at identifying the genetic basis of AIC in an international cohort of 19 patients. After excluding a skewed X chromosome inactivation and the presence of chromosomal rearrangements, I performed WES in trios. The analysis of the data from WES did not allow me to identify any recurrent variants. I therefore tested a new approach combining Whole Genome Sequencing (WGS) and RNA-Sequencing (RNA-Seq) on fibroblast cells. I identified a number of deregulated transcripts implicated in brain and eye development. I compared the results of this analysis with the WGS analysis in order to find variants in these candidate genes. In conclusion, these studies have improved the knowledge of the molecular basis of complex MCD, such as TLE1 in postnatal microcephaly, and revealed the pathogenic mechanisms such as WDR81 in cell cycle progression and EPG5 in endosomes and autophagy. My work has also generated a collection of NGS data (WES, WGS and RNA-Seq) that will be shared in an international consortium to develop new analytical strategies, in particular for the non-coding DNA regions. This novel strategy provides opportunities to improve understanding of the cellular mechanisms involved in brain and eye development
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Plantier, Jean-Luc. "La thrombine dans la physiopathologie vasculaire : une étude structure-fonction". Université Joseph Fourier (Grenoble), 1994. http://www.theses.fr/1994GRE10179.
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La thrombine est une glycoproteine de la famille des proteases a serine. Comparee a celles des autres membres de sa famille, sa structure tertiaire possede une serie de boucles d'insertion particulierement exposees, organisees autour du site actif. Parce que la specificite restreinte de la thrombine pour ses substrats semble faire appel a des interactions a la surface de la molecule, il nous est apparu interessant de determiner quels etaient les roles de ces boucles dans certaines des activites de l'enzyme. Des mutations ponctuelles ont ete realisees dans deux de ces boucles (la boucle b et la boucle ) ainsi que sur la serine du site actif. L'analyse des resultats nous a permis de conclure qu'une thrombine dont le site actif est mutee est denuee de toute activite catalytique. La boucle b est confirmee dans son role de determinant majeur lors de la reconnaissance de tous les substrats testes. En revanche, les mutants que nous avons realises dans la boucle ne modifient que subtilement la poche de specificite primaire et peu les interactions avec les substrats macromoleculaires. En plus de cette etude structurale, nous avons etudier le mecanisme d'augmentation de la permeabilite de l'endothelium induite par la thrombine. La variation de permeabilite depend exclusivement du clivage du recepteur et necessite donc une enzyme catalytiquement active. La reponse des cellules endotheliales depend aussi de l'activation de la proteine kinase c et de phosphorylation sur les tyrosines. Le retour d'une sensibilite a l'enzyme requiere la synthese de nouveaux recepteurs. L'utilisation de nos outils moleculaires nous a permis de montrer que l'activite catalytique de l'enzyme est necessaire pour activer les megacaryocytes et inhiber specifiquement la pousse de leurs progeniteurs, ces deux mecanismes etant medies par le recepteur de la thrombine
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Kim, Kyung-Sun. "Mutation, migration, dissémination dans le travail in situ". Paris 1, 2002. http://www.theses.fr/2002PA010656.
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C'est à un grand voyage que je convie le lecteur à travers ma recherche, entre deux continents, l'Orient et l'Occident. Mais c'est aussi d'un cheminement labyrinthique dont j'essaie de témoigner. Cheminement au cours duquel j'ai fait des rencontres inattendues et des découvertes surprenantes sur moi-même. Cet aller et retour continuel entre la France et la Corée, a engendré puis alimenté des pratiques artistiques nouvelles. N me fallait parler ici et là-bas, de trois éléments symboliques. L'algue, le papier et la grue. Au-delà de la langue, c'est à travers l'espace plastique que j'ai choisi de m'exprimer. Tous ces déplacements et tous ces changements de lieux au sens propre, s'accompagnent de transformations multiples qui touchent ma pratique Jusque dans la conception du rapport au public. Le travail in situ, axe essentiel de cette réflexion. Serait-1l le point de convergence de trois éléments, la mutation, la migration et la dissémination ? Cette recherche tentera d'y répondre.
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Eöry, Lél. "Inferring strength of selection in vertebrate genomes". Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/4925.
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Protein-coding sequences have long been assumed to evolve under selection, but the quantification of the process at the nucleotide sequence level only started when a simple null model, the neutral theory of molecular evolution, was formulated by Kimura. Several methods were developed, which were based on the assumption that synonymous sites (nucleotides at third codon positions which do not change the encoded amino acid) evolve close to neutrally, and could be used as local neutral standards. Most of our current knowledge on the direction and strength of selection still depends on this simple assumption. One method, notably the non-synonymous to synonymous substitution rate ratio (dN/dS) has gained prevalence and is still widely used, in spite of the growing body of evidence that synonymous sites evolve under selection. In this thesis, I quantify the strength of selection in different sequence compartments of mammalian genomes, in order to obtain estimates of their functional importance from comparative genomics analyses. I quantify the fraction of mutations that have been selectively eliminated since the divergence of the species pairs examined, the so called genome wide selective constraint. This in turn is used to approximate the genomic deleterious mutation rate, which is an important parameter for several evolutionary problems. As estimates of selection depend on a large extent on the chosen neutral standard, here I use orthologous transposable elements, so called ancestral repeats, as these have been found to be evolving at a largely neutral fashion, and contain the least number of constrained sites in mammalian genomes. This enables me to quantify the level of selection even at synonymous sites, and the results suggest that these sites indeed evolve under constraint, the consequences of which I discuss. The selective constraint estimates enable me to test some simple hypotheses, such as Ohta's nearly neutral theory of molecular evolution, which suggests that selection is more efficient in species with larger effective population sizes. Beside the choice of neutral standards, there are several additional factors which are known to affect the selective constraint estimates. Here I also test the consequences of one of these, notably when sequences are not at compositional equilibrium (i.e. their GC content is away from the equilibrium GC content), which predicts that sequences with different GC content should evolve with different rates. This can cause bias in the estimates of level of selection or can even imitate selection in sequences which evolve completely neutrally. This effect is quantified here, and a simple correction is discussed.
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Chen, Wei 1965. "Site Directed Mutagenesis Of Dienelactone Hydrolase". Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc500900/.
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The role of individual amino acid residues of the enzyme dienelactone hydrolase was investigated. Using the polymerase chain reaction (PCR), a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHI to EcoRI clcD subclone in the plasmid pUC19. Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. Specifically modified proteins from mutated clcD genes (Arg 81 to alanine, Tyr 85 to phenylalanine and Arg 206 to alanine), were encoded by the mutant clones. Enzyme assays showed that dienelactone hydrolase activity of the mutants Arg 81 and Arg 206 was totally abolished. The DLHase enzyme activity of mutant Tyr 85 is greatly decreased by approximately two thirds.
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Kiflemariam, Sara. "Expression and Mutation Analyses of Candidate Cancer Genes In Situ". Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-184510.
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Cancers display heterogeneity in genetic profiles of the individual cancer cells and in the composition of different malignant and non-malignant cell populations. Such intra-tumor heterogeneity plays a role in treatment response and the emergence of resistance to cancer therapies. Approaches that address this complexity and improve stratification of patients for treatment are therefore highly warranted. Thus, the aims of this thesis were to further develop and apply in situ technologies for expression and mutation analyses of candidate cancer genes to gain a deeper understanding of cancer biology and to study intra-tumor heterogeneity. In paper I, we established and validated a procedure for scalable in situ hybridization of large gene sets in human formalin-fixed paraffin-embedded tissues for analysis of gene expression. This method was used in paper II for large-scale expression analysis of the tyrosine kinome and phosphatome, two gene families whose members are frequently mutated in many forms of cancers. Systematic, compartment-specific expression mapping at cell type resolution enabled us to identify several novel vascular markers that have gone unnoticed in bulk transcriptomic analyses. In papers III and IV, we used padlock probes for in situ mutation detection in single cells for studies of genetic intra-tumor heterogeneity. In paper III, multiplex detection and genotyping of oncogenic point mutations was demonstrated in routinely processed tissue materials, whereas in paper IV we further the application by demonstrating multiplex detection of fusion gene transcripts. Collectively, the work presented in this thesis employs in situ-based methods to obtain spatial resolution of gene expression and mutation patterns in normal and cancer tissues, thereby broadening our understanding of the cancer genome.
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Rehmani, Imran J. "Studying the DNA Binding and Conformation of Metal-Binding Site Mutations in Pirin". Digital Archive @ GSU, 2012. http://digitalarchive.gsu.edu/chemistry_theses/53.
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The transcription factor NF-κB interacts with many other co-regulator proteins that modulate its binding and transcriptional activity. One of these co-regulators, Pirin, is an iron-dependent metalloprotein that has been shown to enhance the DNA binding of NF-κB homodimers. Here, we characterize the interactions between Pirin and its known NF-κB binding partners and examined the role of Bcl-3, a protein that is required for Pirin’s interaction with p50. In addition, we use site-directed mutagenesis to alter conserved residues within Pirin’s metal binding environment and observed how it affected the DNA binding and conformation of the Pirin-NF-κB complex. These studies show that, while a similar enhancing effect on DNA binding is observed, the interactions of Pirin with different NF-κB members are distinct from each other and could possibly have different physiological purposes.
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Mathu, Alexander Muchugia Nganga. "Structural analysis of effects of mutations on HIV-1 subtype C protease active site". Thesis, Rhodes University, 2012. http://hdl.handle.net/10962/d1004073.
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HIV/AIDS is a global pandemic that poses a great threat especially in Sub-Saharan Africa where the highest population of those infected with the virus is found. It has far reaching medical, socio-economic and scientific implications. The HIV-1 protease enzyme is a prime therapeutic target that has been exploited in an effort to reduce morbidity and mortality. However problems arise from drug toxicity and drug-resistant mutations of the protease which is a motivation for research for new, safer and effective therapies. Evidence exists to show that there are significant genomic differences in Subtype B and C that have a negative effect on the intrinsic binding of inhibitors. It is imperative to look at all perspectives from epidemiological, molecular to the pharmacological ones so as to achieve rational design of therapeutic agents. This study involved the use of in silico structural analysis of the effects of mutations in the active site. The data was provided by the National Institute of Communicable Diseases consisting of HIV-1 Subtype C protease sequences of 29 infants exhibiting drug-resistance to ritonavir and lopinavir. The major active site mutations causing drug resistance identified in this study were M46I, I54V and V82A using the Stanford HIV database tool. Homology modeling without extra restraints produced models with improved quality in comparison to those with restraints. MetaMQAPII results differed when models were visualized as dimers giving erroneous modeled regions in comparison to monomers. A broader study with a larger dataset of HIV-1 subtype C protease sequences is required to increase statistical confidence and in order to identify the pattern of drug resistant mutations. Homology modeling without extra restraints is preferred for calculating homology models for the HIV-1 subtype C. Further investigations needs to be done to ascertain the accuracy of validation results for dimers from MetaMQAPII as it is designed for evaluation of monomers.
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Sundström, Hannah. "Mutation and Diversity in Avian Sex Chromosomes". Doctoral thesis, Uppsala University, Department of Evolutionary Biology, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3732.
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Sex chromosomes are useful for the study of how factors such as mutation, selection, recombination and effective population size affect diversity and divergence.
A comparison of gametologous introns in seven different bird species revealed a complete lack of diversity on the female-specific W chromosome. In contrast, Z had at least one segregating site in all examined species. This can be explained by the lower mutation rate and lower effective population size of W but also suggests that selection affects diversity levels on the non-recombining W chromosome.
In a diverse set of chicken breeds, the Z chromosome showed reduced diversity compared to autosomes and significant heterogeneity in levels of variation. High variance in male reproductive success, leading to a reduced Z chromosome effective population size, can partly explain this observation. In addition, we suggest that selective sweeps frequently act on the Z chromosome and are responsible for a significant part of the observed Z reduction.
Differences in the mutation rate of Z and W chromosome sequences indicate that the time spent in male germ line is important for the mutation rate, but does not exclude a specifically reduced mutation rate on the Z chromosome. Estimates of mutation rate in autosomal, Z- and W-linked chicken and turkey sequences indicate a slight reduction in the rate on Z. However, due to rate heterogeneity among introns this reduction is not significant and we cannot exclude male biased mutation as the single cause of rate variation between the chromosomal classes.
Analysis of indel mutation rates in avian and mammalian gametologous introns show frequent occurrence of indels on both W and Y, excluding meiotic recombination as the only source of this type of mutation. The different indel rate patterns in birds (Z>W) and mammals (X=Y) suggest that indels are caused by both replication and recombination.
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FLORENTZ, EGELE CATHERINE. "L'extremite 3'oh aminoacyable du rna du virus de la mosaique jaune du navet : relations entre structure et fonctions". Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13181.
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Analyse structurale (structure secondaire et tertiaire) d'un fragment contenant la sequence "trna like" (extremite 3' oh possedant plusieurs caracteristiques d'un arn de transfert). Modelisation de la structure sur ecran graphique. Analyse des zones de contact entre ce fragment d'arn et la valyl-trna synthetase, a l'aide de differentes sondes structurales. Discussion sur le role de l'extremite "trna like" dans le cycle de developpement du virus (regulation de la traduction de l'information genetique portee par l'arn viral)
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Devaiah, Shivakumar P., i Cecelia A. McIntosh. "Site-Directed Mutational Analysis of Flavonol 3-0-Glucosyltransferases from Citrus paradisi". Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etsu-works/340.
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Glucosyltransferases (GTs) are the important group of enzymes which facilitates the incorporation of UDPactivated glucose to a corresponding acceptor molecule through glucosylation. Glucosylation is a common alteration reaction in plant metabolism and is regularly associated with the production of secondary metabolites. Glucosylation serves a number of roles within metabolism including: stabilizing structures, affecting solubility, transport, and regulating the bioavailability of the compounds for other metabolic processes. GTs involved in secondary metabolism share a conserved 44 amino acid residue motif (60–80% identity) known as the plant secondary product glucosyltransferase (PSPG) box, which has been demonstrated to include the UDP-sugar binding moiety. Among the secondary metabolites, flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. Custom design of enzymes requires understanding of structure/function of the protein. The present study focuses on creating mutant Flavonol- 3-O- Glucosyltransferases proteins using site-directed mutational analysis and testing the effect of each mutation on substrate specificity and kinetic properties of the enzyme.
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Grundberg, Ida. "Genotyping and Mutation Detection In Situ : Development and application of single-molecule techniques". Doctoral thesis, Uppsala universitet, Molekylära verktyg, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-149776.
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The human body is composed of trillions of cells closely working together to maintain a functional organism. Every cell is unique in molecular composition and can acquire genetic variations that might cause it to turn pathological. It is essential to develop improved tools to better understand the development of normal and disease tissue, ideally enabling single-cell expression studies in preserved context of complex tissue with single-nucleotide resolution. This thesis presents the development and application of a new in situ method for localized detection and genotyping of individual transcripts directly in cells and tissues. The described technique utilizes padlock probes and target-primed rolling circle amplification and is highly suitable for sensitive in situ analysis. First, a new strategy for directed cleavage of single stranded DNA was investigated, e.g. nucleic acid targets with extended 3´ ends, for successful initiation of rolling circle amplification. The presented cleavage strategy is simple and applicable for subsequent enzymatic reactions, e.g. ligation and polymerization. Specific cleavage of long target overhangs was demonstrated in synthetic oligonucleotides and in genomic DNA and the detection efficiency was substantially increased. For multiplex detection and genotyping of individual transcripts in single cells, a new in situ method was developed. The technique showed a satisfactorily detection efficiency and was later applied as a general mutation analysis tool for detection of KRAS point mutations in complex tumor tissue sections, e.g. formalin-fixed, paraffin-embedded tumor tissues and cytologic tumor imprints. Mutation status was assessed in patient samples by in situ padlock probe detection and results were confirmed by DNA-sequencing. Finally, the method was adapted for simultaneous detection of individual mRNA molecules and endogenous protein modifications in single cells using padlock probes and in situ PLA. This assay will be useful for gene expression analysis and exploration of new drugs with vague effector sites. To our knowledge, no other technique exists today that offers in situ transcript detection with single-nucleotide resolution in heterogeneous tissues. The method will especially be suitable for discrimination of highly similar transcripts, e.g. splice variants, SNPs and point mutations, within gene expression studies and for cancer diagnostics.
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Sanders, Stephen Anthony. "The effect of selected active site mutations on the properties of horseradish peroxidase isoenzyme C". Thesis, University of Sussex, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.357651.
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Hersh, Megan N. "VISUALIZING GENOMIC INSTABILITY: IN SITU DETECTION AND QUANTIFICATION OF MUTATION IN MICE". University of Cincinnati / OhioLINK, 2001. http://rave.ohiolink.edu/etdc/view?acc_num=ucin991312483.
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Schmitz, Stephan. "Mutational analysis of proposed myosin binding sites on actin". Thesis, University of York, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301148.
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Ajlani, Ghada. "Détermination des sites de mutation responsables de résistance aux herbicides chez des mutants de la cyanobactérie Synechocystis PCC 6714 : étude de l'effet de ces mutations sur le transfert d’électrons du photosystème II". Paris 11, 1989. http://www.theses.fr/1989PA112130.
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Li, Zhen. "Structural and functional consequences of single mutations at the high affinity binding site of cyanovirin-N". Diss., University of Iowa, 2016. https://ir.uiowa.edu/etd/3133.
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This thesis focuses mainly on the consequences that single mutations have on structural, functional and energetic aspects of the protein cyanovirin-N. In order to estimate the free energy of single mutations, we have applied thermodynamics integration and Bennett acceptance ratio techniques. Replica exchange molecular dynamics has been applied to accelerate simulations for complicated scenarios. Our studies suggest that certain single mutations may be promising to improve binding affinity to Manα1→2Manα but we also learned that the simplistic view of a strong hydrogen bond correlating to a high binding affinity may not always be correct. Finally, we explored in detail the widely used mutation P51G for its impact on protein rigidity at the very important hinge region as well as for its possible effect on glycan binding.
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Crandall, Jacob N. "Ribosomal RNA Mutations that Inhibit the Activity of Transfer-Messenger RNA of Stalled Ribosomes". Diss., CLICK HERE for online access, 2010. http://contentdm.lib.byu.edu/ETD/image/etd3535.pdf.
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Suleman, Essa. "Mutational analysis of the PacC binding sites within the aflR promoter in Aspergillus flavus". Thesis, Nelson Mandela Metropolitan University, 2011. http://hdl.handle.net/10948/d1012683.
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It is generally known that media containing simple sugars (sucrose, glucose) and organic nitrogen sources (ammonium) when buffered to acidic pH stimulates aflatoxin production in Aspergillus flavus & A. parasiticus while lactose, nitrate and an alkaline pH inhibit aflatoxin biosynthesis. It has been shown that pH of the growth medium is the most important regulatory factor for aflatoxin biosynthesis since media containing stimulatory carbon and/or nitrogen sources (sucrose and ammonia) do not enhance aflatoxin (or sterigmatocystin) production at alkaline pH. RNA interference (in A. flavus) of the pH regulatory transcription factor, PacC, resulted in aflatoxin production under acidic and alkaline pH conditions whilst wildtype Aspergillus flavus produced aflatoxins only under acidic conditions. This conclusively proved that PacC negatively regulates aflatoxin production at alkaline pH in A. flavus. However the exact mechanism involved in PacC repression of aflatoxin biosynthesis at alkaline pH still remains unknown. The AflR protein is essential for expression of several genes in the aflatoxin biosynthetic cluster. In the current study, sequence analysis of the aflR promoter indicated the presence of two putative PacC binding sites within the aflR promoter of A. flavus 3357WT located at positions -162 and -487 bp from the start codon. The presence of the PacC binding sites in the aflR promoter indicated a possible link between aflR expression and PacC regulation under alkaline conditions. Thus, in this study, it was hypothesized that at alkaline pH, PacC inhibits aflR expression by binding to one or both of the PacC binding sites within the aflR promoter. This in turn, would result in inhibition of aflatoxin biosynthesis since expression of several aflatoxin biosynthetic pathway genes is dependent on activation by AflR. The aim and objective of this study was to test the validity of this hypothesis i.e. that at alkaline pH PacC binds to one or both of its recognition sites within the aflR promoter thereby inhibiting aflR expression which subsequently would result in inhibition of aflatoxin biosynthesis. This was done by first mutating each individual and then both PacC binding sites in the A. flavus 3357 aflR promoter via Single-Joint PCR (SJ-PCR) and fusing the wildtype and each mutated aflR promoter to the Green Fluorescent Protein (gfp) gene and the trpC terminator to yield a functional expression vector. These constructs were then transformed into A. flavus 3357.5. Positive transformants were confirmed to express GFP by fluorescence microscopy and spectrofluorometry. Quantification of GFP protein levels of the various transformants in this study indicated that PacC negatively regulated aflR promoter activity at alkaline pH. RT-qPCR was performed on positive transformants after growth on SLS medium at acidic and alkaline pH to determine if PacC negatively regulated aflR promoter activity at alkaline pH and to determine whether PacC binds preferentially to one or both recognition sites within the aflR promoter. RT-qPCR analysis suggest that PacC binds non-preferentially to both recognition sites within the aflR promoter on sucrose and lactose media at alkaline pH, although mutation of PacC binding site 2 results in a slightly higher expression compared to mutation of PacC binding site 1. Increasing the concentration of an aflatoxin conducive nitrogen source stimulated aflR promoter activity but this was not sufficient to overcome negative regulation by PacC. It is generally known that repression of aflR expression results in repression of aflatoxin biosynthesis irrespective of pH. The results of this study strongly suggest that PacC negatively regulates aflR promoter activity at alkaline pH by binding to one or both PacC recognition sites within the aflR promoter. Since aflR promoter activity is repressed by PacC at alkaline pH, this substantiates the hypothesis that PacC represses aflatoxin biosynthesis by inhibiting expression of aflR. Furthermore, the results of this study indicated that there may be some PacC protein present in the active form at acidic pH irrespective of the carbon source and nitrogen source used in the growth medium. RT-qPCR analysis indicated that any active PacC present at acidic pH may cause repression of the aflR promoter based on the position of the PacC binding site relative to the aflR start codon, although it appears that PacC may have a higher affinity for PacC binding site 2 (which is closer to the aflR start codon).
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Beal, Brian D. "UV-induced mutations at a non-dimer site in E. coli : possible role for a TA* photoproduct /". Available to subscribers only, 2007. http://proquest.umi.com/pqdweb?did=1400950891&sid=1&Fmt=2&clientId=1509&RQT=309&VName=PQD.
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Thesis (M.S.)--Southern Illinois University Carbondale, 2007."Department of Molecular Biology, Microbiology and Biochemistry." Includes bibliographical references (leaves 67-74). Also available online.
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Seedy, Ayman Salah Ahmed El. "Études moléculaire et cellulaire des mutations du gène CFTR : de la génétique à la fonctionnalité de la protéine". Poitiers, 2011. http://nuxeo.edel.univ-poitiers.fr/nuxeo/site/esupversions/1f04df89-c6ec-4a24-a424-49e80e1ea1a4.
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La mucoviscidose est la maladie génétique grave à transmission autosomique récessive la plus fréquente dans les populations d'origine européenne. Cette pathologie est due au dysfonctionnement de la protéine CFTR (Cystic Fibrosis Transmembrane conductance Regulator), canal chlorure présent à la membrane apicale des cellules épithéliales. La gravité de la pathologie dépend de la ou des mutations du gène CFTR. L’objectif de nos travaux est de comprendre l’impact des mutations du gène CFTR afin d’établir un conseil génétique avisé. Pour cela, nous avons dans un premier temps déterminé quelles sont les mutations présentes sur l'exon 9 du gène. A cause de la présence de copies de cette région, nous avons établi de nouvelles conditions expérimentales pour étudier exclusivement le gène, et ainsi deux pseudomutations ont été détectées. Dans un second temps, nous avons étudié trois allèles complexes contenant des mutations fréquentes de CFTR (D443Y, G576A, R668C) et une mutation rare, G149R. In vitro nous avons mis en évidence l’effet délétère de G149R seule ou en complexe, et la diminution de la quantité de la protéine mature lorsque les autres allèles complexes sont présents. Dans la dernière partie de ce travail, nous avons mis en place au laboratoire les techniques d’étude de l’épissage. Pour cela, nous avons construit un minigène hybride qui nous permet de définir le rôle exact des substitutions nucléotidiques sur l'épissage alternatif. Les résultats obtenus à partir de ces trois études nous ont permis de mieux comprendre l'impact des mutations étudiées et ainsi d'émettre un diagnostic moléculaire documentéCystic fibrosis is a serious genetic disease autosomal recessive most frequent in populations of European origin. This pathology is due to dysfunction of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) chloride channel present in the apical membrane of epithelial cells. The severity of the disease depends on mutations in the CFTR gene. The objective of our work is to understand the impact of CFTR mutations in order to establish a genetic counseling notified. For this, we initially determined which mutations are present in exon 9 and its flanking regions of the gene. As this region is duplicated in the genome, we established new experimental conditions to study exclusively the gene, and thus two pseudomutations were detected. In the second step, we examined complexes containing three frequent CFTR mutations (D443Y, G576A, R668C) and a rare mutation, G149R. In vitro we have demonstrated the deleterious effect of G149R alone or in complex and the decrease the amount of the mature protein complex when other alleles are present. In the last part of this work, we developed the laboratory techniques for the study of splicing. For this, we constructed a minigene hybrid that allows us to define the exact role of nucleotide substitutions on splicing. The results obtained from these three studies allowed us to better understand the impact of the studied mutations and thus to make a molecular diagnosis documented
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Kergourlay, Virginie. "Mise au point d'outils novateurs pour l'identification de mutations pathogènes : le cas des dysferlinopathies". Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM5044.
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Le diagnostic des maladies génétiques est difficile à émettre. En effet, il est souvent difficile de déterminer comment une mutation va entrainer la pathologie. Le but de cette thèse est de développer des outils permettant de répondre à cette interrogation. Les mutations peuvent entrainer des anomalies à différents niveaux, différentes outils ont ainsi été développées en parallèle afin de pouvoir détecter différents types d'anomalies. Ces outils ont été développés en utilisant comme modèle une maladie génétique appartenant à la famille des myopathies, entrainant une dégénérescence des muscles des patients. Les travaux de cette thèse ont permis de confirmer le caractère délétère de certaines mutations en démontrant des anomalies dans un mécanisme appelé « épissage » qui permet la transmission de l'information contenue dans le génome. Les mutations en empêchant cette transmission vont ainsi être responsables de la maladieDiagnosis of genetic diseases is a difficult task. Indeed, it is often difficult to determine if mutations detected in patients will be responsible of the disease. The aim of this thesis is to develop tools allowing answering on this question. Mutations can have deleterious effects to several levels, thus different tools have been develop in parallel in order to detect different kind of abnormalities. These tools have been developed using as model a genetic disease belonging to the family of myopathy, leading to a degeneration of patients muscles. These thesis works have confirmed the deleterious effect on some mutations in a mechanism named "splicing" which allow transmission of the genome's information. Mutations preventing the transmission will thus be responsible of the disease
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Fabrega, Sylvie. "Glycosyl hydrolases impliquées dans des maladies lysosomales : Analyses de mutations du site actif basées sur des prédiction structurales". Paris, CNAM, 2002. http://www.theses.fr/2002CNAM0433.
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Des glycoside hydrolases (EC. 3. 2. 1. X) (GH) lysosomales dont la fonction consiste en l'hydrolyse de composés glycosylés cellulaires sont impliquées dans des maladies génétiques humaines regroupées sous le terme de "maladies lysosomales". Nous avons effectué des études structurales et fonctionnelles sur certaines de ces GH lysosomales afin de mieux comprendre la physiopathologie des maladies de surcharge métabolique très invalidantes les impliquants. Une vaste étude sur différentes GH, utilisant un même mécanisme catalytique (rétention de la configuration anomérique), incluant des GH lysosomales, a été menée en collaboration avec des spécialistes de modélisation moléculaire. L'analyse de la séquence primaire des protéines par la méthode HCA (Hydrophobic Cluster Analysis) de prédiction 2D, et l'exploitation des banques de donnnées sur des GH cristallisées, ont conduit à la proposition d'un modèle structural de type tonneau (α/ß)8, commun à toutes les enzymes dans le clan des brins ß4 et ß7 du tonneau ont été localisés. Les éléments structuraux et fonctionnels du site catalytique de cinq GH lysosomales : la glucocérébrosidase (maladie de gaucher), l'α-L-iduronidase (mucopolysacharidose de type I), la ß-galactosidase (ganglyosidose GM1), la ß-glucuronidase (syndrome de Sly) et a ß-mannosidase (ß-mannosidose) ont été caractérisés. L'impact structural et fonctionnel de mutations génétiques a été analysé. Nous avons ensuite exploité les prédictions de modélisation effectuées sur ces enzymes pour mieux caractériser les acides aminés, catalyseurs acide-bases et nucléophile, de la glucocérébrosidase et l'α-L-iduronidase humaine ; des protéines pour lesquelles une thérapie enzymatique est développée. A l'aide d'une stratégie de biologie cellulaire basée sur une mutagénèse substitutive des acides aminés suspectés et une expression des protéines recombinantes, mutées et non mutées, dans un système cellulaire hétérologue, nous avons obtenu une invalidation totale de l'activité catalytique des enzymes. Nous avons montré que la perte d'activité n'était pas liée à un défaut de biosynthèse et de maturation des protéines mutées, mais des mutations introduites. Aussi, nous avons été en mesure d'impliquer très fortement les acides glutamiques 235 et 340 de glucocérébrosidase et les acides glutamiques 182 et 299 de l'α-L-iduronidase dans la fonction catalytique de ces GH. Nous avons ainsi apporté une validation expérimentale à nos prédictions. L'ensemble de nos travaux de modélisation moléculaire et d'analyse de relation structure-fonction souligne ici l'importance d'une complémentarité entre les approches de prédictions de structure et les approches de vérifications expérimentales pour la connaissance du fonctionnement catalytique de GH lysosomales impliquées dans des maladies génétiquesGlycosyl hydrolases (GH) are a widespread group of enzymes. Several lysosomal strorage diseases characterized by a severe handicap are due to deficiences in GH. A structural biology study concerning the numerous enzymes belonging to clan GH-A of GH was performed. Clan GH-A includes 5 human enzymes implicated in lysosomal diseases : glucocerebrosidase, α-L-iduronidase, ß-galactosidase, ß-glucuronisade and ß-mannosidase. Predictions concerning the active site structure of these enzymes were made by Hydrophobic Cluster Analysis (HCA), a bidimensional analytical method permitting to compare highly divergent protein sequences. We found that all the active sites may have a similar 3D structures consisting af an (α/β)8 barrel. In particular, a pair of glutamic acid residues, presumed to directly participate in the enzymatic hydrolysis, was identified. Finally, analysis of mutations described in patients was in agreement with the predictions. Next, we performed site-directed mutagenesis studies to obtain experimental evidence supporting our HCA predictions. These studies concerned the glutamatic acid residues supposed to be involved in the catalytic activity of glucocerebrosidase and α-L-iduronidase. Substitution of these glutamatic acids by alanine residues led to complete inactivity of the mutant proteins without affecting their folding/processing. These data further support that Glu235/Glu340 and Glu182/Glu299 play a key role in th enzymatic activity of glucocerebrosidase and α-L-iduronidase, respectively. In conclusion, our work shows that strutural biology and mutagenesis studies may be highly complementary for a better understanding of the structure/function relationship in enzymes involved in severe genetic diseases
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Kang, You-Na. "Studies on the Structure and Function of Soybean β-Amylase by Mutations at Active Site and Surface Residues". Kyoto University, 2004. http://hdl.handle.net/2433/147767.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第10918号
農博第1424号
新制||農||892(附属図書館)
学位論文||H16||N3929(農学部図書室)
UT51-2004-G765
京都大学大学院農学研究科農学専攻
(主査)教授 内海 成, 教授 廣瀬 正明, 助教授 三上 文三
学位規則第4条第1項該当