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1
Reddivari, Muralidhar. "Microbiological biotransformations for drug synthesis." Thesis, University of Ulster, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274094.
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Suen, Yu. "Microbial biosynthesis of neutral lipids." Diss., Georgia Institute of Technology, 1986. http://hdl.handle.net/1853/25197.
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Nakatani, Yoshio, and n/a. "Biochemical characterisation and structural determination of a novel exoglucanase." University of Otago. Department of Biochemistry, 2009. http://adt.otago.ac.nz./public/adt-NZDU20090930.113205.
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Following the successful detection of novel exo-1,3-β-glucanase activity from marine bacterium Pseudoalteromonas sp. BB1 that was isolated from brown algae Durvillaea sp. and its partial gene identification, the exo-glucanase (ExoP) has been purified to homogeneity. Full gene identification of exoP was achieved by Southern hybridisation using a derived probe. In total, 7612 bp of the partial Pseudoalteromonas gDNA sequence was obtained, in which 6 coding regions including the full exoP sequence were identified. The exoP gene consists of 2523 nucleotides, which is translated into 840 amino acids. The first 27 amino acids are predicted to be a signal peptide in agreement with obtained N-terminal sequence of native ExoP. The molecular weight of the mature ExoP portion was calculated to be 89320.5 Da (813 amino acids), consistent with its mobility in SDS-PAGE (87 kDa). Interestingly a putative lichenase gene (licA) whose enzymatic function is related to ExoP was located only 50 bp upstream of exoP suggesting these two genes work co-ordinately. ExoP is classified as a glycosyl hydrolase GH3 family member and is homologous with a group of bacterial and plant enzymes of which barley ExoI is the best characterised.
Following the complete gene identification, exoP was successfully cloned, over-expressed in E. coli and purified. Biochemical characterisation of ExoP revealed that the native and recombinant proteins were identical with optimal temperature 30�C and optimal pH 7.0 for hydrolase activity. ExoP showed substrate specificity towards both 1,3-β- and 1,4-β-glucans but did not hydrolyse aryl substrates unlike other glucosidases in the GH3 family. The ExoP was designated as an exo-1,3/1,4-β-glucanase (EC. 3.2.1-.).
The crystal structure of ExoP was successfully solved at 2.45 Å resolution using a two step molecular replacement procedure. ExoP was found to consist of a distinctive three domain structure: an (α/β)₈ barrel domain A, an (α/β)₆ sheet domain B and a β-sandwich domain X. The catalytic pocket is formed by domains A and B and this two domain structure is highly similar to that of barley exo-1,3/1,4-β-glucanase ExoI.
Three potential subsites were observed in the structure: the -1 subsite that is identical to that of barley ExoI, the +1 subsite that contains an antiparallel tryptophan clamp formed by W294 and W436, and a putative +2 subsite that involves W494. This observation agreed with the prediction by subsite mapping. The function of domain X remains unknown. However it was discovered that this domain is common in marine bacterial GH3 enzymes, and that marine bacteria also produce an independent protein that consists of the C-terminal half of this domain. The analysis of ExoP structure showed not only the conserved features of the -1 and +1 subsites of GH 3 family enzymes but new insights such as the hinge action between domains A and X, mobility of a flexible loop near the catalytic site and a possible role of domain X contributing to the enzyme fidelity.
The second part of this project focused on glycosynthase activity generated by active site mutation. While ExoP showed no such activity the Glu to Ser mutant of exo-1,3-β-glucanase (Exg) from Candida albicans was functional. Albeit native Exg shows high specificity towards 1,3-β-glucans, using a donor 1-fluoro-α-D-glucose (1FG) and an acceptor p-nitrophenyl β-D-glucopyranoside (pNPG) the mutant E292S-Exg glycosynthase preferentially forms a 1,6-β-linked product. In this study, the crystal structure of E292S-Exg complexed with p-nitrophenyl β-gentiobioside (pNPgent), E292S-Exg/pNPgent (the product formed by the above reaction) was solved at 1.60 Å. Comparison of this structure with the previously solved complexed structure, E292S-Exg/1FG/pNPG did not explain why the 1,6-linkage was favoured by this enzyme but surprisingly revealed movement of glucose at the -1 subsite, which is organised by a complex hydrogen bond network, but did not show movement of glucose in the phenylalanine clamp (the +1 subsite). The presence of an aromatic clamp (Phe-Phe in Exg or Trp-Trp as seen in ExoP) in an exo-glucanase is seen to contribute to specificity but not explain it. Glycosynthesis using other acceptor oligosaccharides was also explored in this study. Exg glycosynthase showed broad specificity using p-nitrophenyl derivatised mono-saccharides. However, it remains unknown whether this broad specificity is acceptor dependent or intrinsically due to the mutation created in Exg.
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Tabarya, Daniel. "Studies of the Membrane and DNA Gyrase Inhibiting Antibiotics on Pigment Synthesis in Corynebacterium Poinsettiae." Thesis, University of North Texas, 1988. https://digital.library.unt.edu/ark:/67531/metadc935771/.
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The purpose of this study was (1) to determine whether a correlation exists among the protein profiles, extracted from cell membranes of mutants belonging to five pigment cluster groups, (2) to locate the protein moiety and cartenoprotein complex in the membranes of wild type and colorless mutant (designated W-19) of C. poinsettae and to show whether there are any structural differences between cell membranes of the wild type and a colorless mutant, (3) to determine the effect of six antibiotics on cartenoid gene expression.
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Wyk, Paul. "Genetics of abequose biosynthesis in the rfb region of Salmonella typhimurium LT2 /." Title page, contents and abstract only, 1988. http://web4.library.adelaide.edu.au/theses/09PH/09phw981.pdf.
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McClanahan, Robert Henry. "Microbial and chemical transformations of cannabinoids and related alkyl phenols /." The Ohio State University, 1985. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487261919113874.
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Clarke, Kim Gail. "A reassessment of the production of acetone and butanol by Clostridium acetobutylicum in continuous culture." Doctoral thesis, University of Cape Town, 1987. http://hdl.handle.net/11427/21918.
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Bibliography: pages 154-195.The production of acetone and butanol by Clostridium acetobutylicum P 262 was studied in continuous culture under conditions where the nutrients were present in excess of the requirements and the cell growth was limited by the products formed during the fermentation. This system differs from most continuous culture systems used to obtain solvent production where the limitation of a specific nutrient was utilised to limit the cell growth.
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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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Brewster, Rachel Elizabeth. "Synthesis of small molecules with specific function : I. Peptidocalix[4]arenes as molecular receptors ; II. Towards the total synthesis of (-)-Dihydroguaiaretic acid." Diss., Available online, Georgia Institute of Technology, 2004:, 2004. http://etd.gatech.edu/theses/available/etd-06072004-131103/unrestricted/brewster%5Frachel%5Fe%5F200405%5Fphd.pdf.
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Lau, Man Kit. "Syntheses and downstream purification of 1,2,4-butanetriol." Diss., Connect to online resource - MSU authorized users, 2007.
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Jancauskas, Justas. "Strategies for improving synthesis of quinic acid and shikimic acid from D-glucose." Diss., Connect to online resource - MSU authorized users, 2008.
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Bongat, Aileen Fay Galos. "Synthesis and Evaluation of Structural Analogues of Escherichia coli Lipid A for Application Towards CD14-Targeting Glycotherapeutics." Diss., St. Louis, Mo. : University of Missouri--St. Louis, 2008. http://etd.umsl.edu/r3301.
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Martinez, Robert J. "Multiscale analyses of microbial populations in extreme environments." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/24754.
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Thesis (Ph.D.)--Biology, Georgia Institute of Technology, 2008.Committee Chair: Patricia Sobecky; Committee Member: Ellery Ingall; Committee Member: Jim Spain; Committee Member: Martial Taillefert; Committee Member: Thomas DiChristina.
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Shipley, Paul R. "The biosynthesis of the thiopeptide antibiotic thiostrepton /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/11559.
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Davis, Kelley L. "Mycoplasma fermentans MALP-404 : a new paradigm for surface variation of mycoplasmas /." Free to MU Campus, others may purchase, 2003. http://wwwlib.umi.com/cr/mo/fullcit?p3091915.
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Vangala, Lakshmisri Manisha. "Size Dependent Antimicrobial Properties of Sugar Encapsulated Gold Nanoparticles." TopSCHOLAR®, 2012. http://digitalcommons.wku.edu/theses/1166.
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The antimicrobial properties of dextrose encapsulated gold nanoparticles (dGNPs) with average diameters of 25 nm, 60 nm, and 120 nm (± 5 nm) synthesized by green chemistry principles were investigated against both Gram-negative and Gram-positive bacteria. Studies were performed involving the effect of the dGNPs on the growth, morphology and the ultrastructural properties of bacteria. dGNPs were found to have significant dose dependent antibacterial activity which was directly proportional to their size and also their concentration. The microbial assays revealed the dGNPs to be bacteriostatic as well as bactericidal. The dGNPs exhibited their bactericidal action through the disruption of the bacterial cell membrane causing leakage of cytoplasmic content. The overall outcomes of this study suggest that dGNPs hold promise as a potent antimicrobial agent against a wide range of disease causing bacteria and can control and prevent possible infections or diseases.
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Schmelz, Stefan. "Adenylate forming enzymes involved in NRPS-independent siderophore biosynthesis." Thesis, University of St Andrews, 2010. http://hdl.handle.net/10023/997.
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Activation of otherwise unreactive substrates is a common strategy in chemistry and in nature. Adenylate-forming enzymes use adenosine monophosphate to activate the hydroxyl of their carboxylic substrate, creating a better leaving group. In a second step this reactive group is replaced in a nucleophilic elimination reaction to form esters, amides or thioesters. Recent studies have revealed that NRPS- independent siderophore (NIS) synthetases are also adenylate-forming enzymes, but are not included in the current superfamily description. NIS enzymes are involved in biosynthesis of high-affinity iron chelators which are used for iron acquisition by many pathogenic microorganisms. This is an important area of study, not only for potential therapeutic intervention, but also to illuminate new enzyme chemistries. Here the structural and biochemical studies of AcsD from Pectobacterium chrysanthemi are reported. AcsD is a NIS synthetase involved in achromobactin biosynthesis. The co-complex structures of ATP and citrate provide a mechanism for the stereospecific formation of an enzyme-bound citryl-adenylate. This intermediate reacts with L-serine to form a likely achromobactin precursor. A detailed characterization of AcsD nucleophile profile showed that it can not only catalyze ester formation, but also amide and possibly thioester formation, creating new stereospecific citric acid derivatives. The structure of a N-citryl-ethylenediamine product co-complex identifies the residues that are important for both recognition of L-serine and for catalyzing ester formation. The structural studies on the processive enzyme AlcC, which is involved in the final step of alcaligin biosynthesis of Bordetella pertussis, show that it has a similar topology to AcsD. It also shows that ATP is coordinated in a manner similar to that seen in AcsD. Biochemical studies of a substrate analogue establish that AlcC is not only capable of synthesizing substrate dimers and trimers, but also able to assemble the respective dimer and trimer macrocycles. A series of docked binding models have been developed to illustrate the likely substrate coordination and the steps along dimerization and macrocyclization formation. Structural and mechanistic comparison of NIS enzymes with other adenylate-forming enzymes highlights the diversity of the fold, active site architecture, and metal coordination that has evolved. Hence, a new classification scheme for adenylate forming enzymes is proposed.
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Chan, K. K. Jason. "Identification and enzyme studies of rare amino acid biosynthesis from Streptomyces cattleya." Thesis, University of St Andrews, 2013. http://hdl.handle.net/10023/4478.
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This thesis is focussed on the biosynthesis of three toxins: fluoroacetate, 4-fluoro-L-theronine and β-ethynyl-L-serine which are biosynthesised by the soil bacteria Streptomyces cattleya. The two fluorinated metabolites originate from a common biosynthetic pathway and the thesis describes studies carried out on an aldose-ketose isomerase enzyme of the pathway. The biosynthetic origin of β-ethynyl-L-serine is not known. A total synthesis of this acetylenic amino acid is descibed along with the development of a new analytical method for identifying the metabolite and for future isotope-labelling based biosynthetic studies. Chapter 1 presents the background of this research. It is focussed on the biosynthesis of fluoroacetate and 4-fluoro-L-threonine by S. cattleya and it also introduces alkyne-containing natural products and their biosynthesis. Chapter 2 describes the work carried out on crystallisation of the aldose-ketose isomerase of the fluorometabolite pathway in S. cattleya. Crystals of the isomerase were obtained and they were diffracted by X-ray, however a structure could not be solved. Chapter 3 contains site-directed mutagenesis studies of the isomerase from S. cattleya. Chapter 4 describes an enantioselective total synthesis of β-ethynyl-L-serine. A robust analytical technique based on derivatisation using 'Click' chemistry and LC-MS was developed for the detection of this amino acid directly from the fermentation broth. Chapter 5 details the experimental procedures for compounds synthesised in this thesis and the biological procedures for gene cloning and protein purification.
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Ksenija, Pavlović. "Mikrotalasno stimulisane transformacije prirodnih i sintetičkih karboksilnih kiselina i njihovih derivata." Phd thesis, Univerzitet u Novom Sadu, Prirodno-matematički fakultet u Novom Sadu, 2014. https://www.cris.uns.ac.rs/record.jsf?recordId=87883&source=NDLTD&language=en.
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Predviđena istraživanja u ovoj doktorskoj disertaciji su usmerena u pravcu modifikacije klasičnih metoda i postupaka za transformaciju karboksilne grupe prirodnih i sintetičkih karboksilnih kiselina. Modifikacije su rađene novim, savremenim, ekonomski i ekološki opravdanim metodama rada u mikrotalasnom reaktoru. Modifikovanim sintetskim postupcima u mikrotalasnom reaktoru urađena je sinteza amida, hidroksamskih derivata, kao i redukcija individualnih naftnih kiselina i smeše prirodnih naftnih kiselina „Velebit“ do alkohola. Prirodne naftne kiseline korišćene u ovom radu su prvo izolovane iz gasne frakcije (interval destilacije 168-290 °C) vojvođanske nafte „Velebit“ a potom prečišćene i razdvojene na uže frakcije na osnovu različite kiselosti. Nakon toga, izvršena je njihova karakterizacija GC-MS-EI analizom (čime je potvrđeno da dolazi do strukturne diferencijacije kiselina). U radu je takođe ispitana biološka aktivnost sintetizovanih derivata. Proučavan je uticaj prirodnih naftnih kiselina „Velebit“ i njenih derivata na rast pet sojeva Pseudomonas sp., kao i uticaj odabranih sintetisanih jedinjenja na proliferaciju četiri ćelijske linije humanih tumora pri čemu je kao kontrola služila jedna zdrava humana ćelijska linija.The investigation of this doctoral dissertation is directed toward the modification of the transformation of the carboxylic group of natural and synthetic carboxylic acids. The dissertation takes into consideration the classical methods and procedures of the reaction and modifies them using microwave reactor. The synthesis of amides, hydroxamic derivatives, as well as the reduction of individual petroleum acids and acid mixtures of natural oil "Velebit" to alcohol were achieved by the modifications made to the synthetic methods in the microwave reactor. The natural oil acids used within this study were first isolated from the gas fraction (distillation interval 168-290 °C) of the Vojvodina's crude oil "Velebit", and then purified and separated by the narrow fractions under the different acidity. After that, their characterisation was made by the GC-MS-EI analysis which confirmed that the structural differentiation of acids had been achieved. Also, the biological activity of the synthesized derivatives are analysed. The impact of natural petroleum acids "Velebit" and its derivatives on the growth of five strains of Pseudomonas sp. was studied, as well as the impact of selected synthesized compounds on the proliferation of four human tumor cell lines wherein one healthy human cell lines used as the control.
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Mirjana, Radanović. "Kompleksi nekih prelaznih metala sa Šifovim bazama aminogvanidina." Phd thesis, Univerzitet u Novom Sadu, Prirodno-matematički fakultet u Novom Sadu, 2015. https://www.cris.uns.ac.rs/record.jsf?recordId=95548&source=NDLTD&language=en.
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U ovoj doktorskoj disertaciji opisane su sinteze novih kompleksa prelaznih metala sa piridoksiliden- (PLAG), odnosno saliciliden-aminogvanidinom (SALAG). Dobijenikompleksi su okarakterisani elementalnom analizom, IR spektrima, konduktometrijskim i magnetnim merenjima, a većina i rendgenskomstrukturnom analizom. Osim toga, dobijene su i nove forme ovih Šifovih baza, i to u vidu monokristala, čime su omogućena ispitivanja njihovih molekulskih i kristalnih struktura, kao i uporedna analiza sa koordinovanim ligandima. Sa PLAG je sintetisano 7 novi kompleksa Cu(II), a pored toga po prvi put suizolovani mono i bis(ligand) kompleksi Fe(III) i Co(III), mono(ligand) kompleksi V(V), kao i jedan kompleks Zn(II) u kojem ovaj potencijalno tridentatni ONN ligand, umonoprotonovanoj formi, ima ulogu kontra-jona. Sa stanovišta geometrije zajedničko za izolovane komplekse Cu(II) i V(V) je da imaju kvadratno-piramidalnu strukturu, sa izuzetkom jednog kvadratno-planarnog kompleksa Cu(II), dok je u kompleksima Fe(III) i Co(III) nađeno očekivano oktaedarsko okruženje centralnogjona. Pored ovih, sintetisano je i pet novih kompleksa sa SALAG, od kojih su dvakompleksa Cu(II) i kompleks V(V) okarakterisani rendgenskom strukturnomanalizom, dok je mikrokristalnim bis(ligand) kompleksima Co(III) i Ni(III) na osnovufizičko-hemijskih karakteristika predložena odgovarajuća struktura. Zajedničko za obe opisane Šifove baze je da se koordinuju na ONN tridentatni način, i to preko atoma kiseonika deprotonovane fenolne grupe i atoma azota azometinske i imino grupe AG fragmeta. Posebno je naglašeno da su saPLAG izolovana dva dimerna kompleksa Cu(II) u kojima je po prvi put nađena tetradentatna koordinacija ovog liganda, u koju je dodatno uključen atom kiseonika hidroksimetil-grupe PL-ostatka. Za razliku od SALAG, koji je u izolovanim kompleksima koordinovan isključivo kao monoanjon, nastao deprotonacijom fenolneOH-grupe, za PLAG je osim ove, potvđena koordinacija u neutralnoj, zwitter-jonskoj, ali i dvostruko deprotonovanoj formi. Zwitter-jonska forma liganda nastaje migracijom atoma vodonika sa fenolnog hidroksila na piridinski atom azota PL-ostatka, dok deprotonacijom piridinskog ili hidrazinskog atoma azota, odnosno oba pomenuta atoma nastaju monoanjon i dianjon helatnog liganda, respektivno. Na kraju, urađena su i ispitivanja antimikrobne aktivnosti odabranih jedinjenjaprema predstavnicima grampozitivnih i gramnegativnih bakterija, kao i dve kulturekvasca. Tom prilikom nije utvrđena nikakva inhibitorna aktivnost prema primenjenimbakterijskim sojevima, dok su u slučaju kvasaca izvesno mikrobicidno dejstvo pokazali samo kompleksi Cu(II).This PhD thesis describes the syntheses of some new transition metal complexes with pyridoxilidene- (PLAG) and salicylideneaminoguanidine (SALAG). Obtained complexes are characterized by elemental analysis, IR spectroscopy, conductometric and magnetic measurements. Besides, the structural analysis of majority of the obtained complexes was performed. Some new forms of these Schiff bases are synthesized in form of single crystals, which made their X-ray analysis as well as comparison with coordinated forms possible.With PLAG, 7 new Cu(II) complexes were obtained and for the first time mono and bis(ligand) complexes of Fe(III) and Co(III) as well mono(ligand) complexes of V(V) were isolated. Furthermore, the structure of Zn(II) complex in which PLAG in its monocationic form has a role of counter ion is presented. With the exception of one Cu(II) complex, all reported Cu(II) and V(V) complexes have a square-pyramidal geometry, whilst Fe(III) and Co(III) are situated in octahedral surroundings. Also, five new complexes of Cu(II), Co(III), Ni(II) and V(V) with SALAG were synthesized. In both Cu(II) complexes and V(V) complex the expected coordination mode and geometry were confirmed by X-ray analysis, while octahedral structure of bis(ligand) complexes with Co(III) and Ni(II) was proposed based on results of physico-chemical characterization.Both PLAG and SALAG coordinate the metal ion in tridentate ONN manner, through the oxygen atom of deprotonated phenolic group and nitrogen atoms of azomethine and imino groups of AG moiety. It is also emphasized that in two dimeric Cu(II) complexes with PLAG tetradentate coordination mode was found, in which the oxygen atom of hydroxymethyl group of PL residue was additionally involved. Unlike SALAG, which is coordinated as monoanion in all of the examined complexes, PLAG can have one of three degrees of deprotonation. Zwitter-ion of PLAG is formed by migration of H-atom from phenolic oxygen to pyridine nitrogen, while the deprotonation of pyridine or/and hydrazine nitrogen, makes it mono-and dianion, respectively.Also, microbiological tests on the selected compounds were preformed. Namely, antimicrobial activity of these compounds against some gram-positive and gram-negative bacteria, as well as some yeast cultures was examined and none of the samples showed antimicrobial activity against bacteria, whilst only Cu(II) complexes showed certain inhibitory effect against yeasts.
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Maria, José Carlos Aires. "Genomic instability associated to impairment of Fe-S clusters synthesis in Saccharomyces cerevisiae yeast cells." Doctoral thesis, Universitat de Lleida, 2014. http://hdl.handle.net/10803/275980.
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Les glutaredoxines (GRXs) són tiol oxidoreductases àmpliament repartides entre organismes procariotes i eucariotes. La Grx5 de llevat Saccharomyces cerevisiae es troba a la matriu mitocondrial i participa en la síntesi dels centres ferro-sofre (ISCs), que són co-factors necessaris per a diversos processos cel•lulars essencials des la respiració, la regulació de l'expressió gènica i el metabolisme de l'ADN-ARN. Algunes proteïnes amb ISCs estan involucrades en el metabolisme de l'ADN, més precisament en la replicació de l'ADN o en els processos de reparació del mateix. Treballs recents van demostrar que els defectes en el metabolisme de ISCs comprometen l'estabilitat del genoma cel•lular, i aquesta inestabilitat està associada a la predisposició a múltiples càncers humans. Mitjançant l'ús d'un mutant Δgrx5 de S. cerevisiae com a model d'estudi vam intentar entendre millor la relació entre la inestabilitat genòmica i els defectes en la biosíntesi de ISCs. L'absència de la proteïna mitocondrial Grx5 condueix a un augment de la inestabilitat genètica. Aquesta inestabilitat del genoma no depèn de l'acumulació de ferro que es produeix en les cèl • lules que no tenen Grx5. Les cèl • lules que no expressen Grx5 tenen majors nivells de dany constitutiu a l'ADN, que s'associa específicament a la formació de "focis" associats a Rad52. Les cèl • lules que no tenen Grx5 i Ssq1 són hipersensibles a agents que danyen l'ADN de forma additiva. Aquesta sensibilitat és independent de l'estat d'estrès oxidatiu constitutiu que es produeix en les cèl • lules que no tenen Grx5. L'absència de les proteïnes Grx5 i Ssq1 provoca un retard en la progressió del cicle cel • lular a través de la fase S. La via de reparació de l'ADN, recombinació homòloga podria tenir un paper important en la reparació dels danys en l'ADN en les cèl • lules del mutant Δgrx5.Las glutaredoxinas (GRXs) son tiol oxidoreductasas ampliamente distribuídas entre organismos procariotas y eucariotas. La proteína Grx5 de levadura Saccharomyces cerevisiae se encuentra en la matriz mitocondrial y participa en la síntesis de los centros hierro-azufre (ISCs), que son co-factores necesarios para varios procesos celulares esenciales, tales como la respiración hasta la regulación de la expresión génica y el metabolismo del ADN-ARN. Algunas proteínas asociadas a ISCs están involucradas en el metabolismo del ADN, más precisamente en su replicación o en procesos de reparación del mismo. Trabajos recientes demostraron que los defectos en el metabolismo de ISCs comprometen la estabilidad del genoma celular, y esta inestabilidad está asociada a la predisposición a múltiples cánceres humanos. Mediante el uso de un mutante Δgrx5 de S. cerevisiae como modelo de estudio quisimos abordar la comprensión de la relación entre la inestabilidad genómica y los defectos en la biosíntesis de ISCs. La ausencia de la proteína mitocondrial Grx5 conduce a un aumento de la inestabilidad genética. Esta inestabilidad del genoma no depende de la acumulación de hierro que se produce en las células que carecen Grx5. Las células que no expresan Grx5 tienen mayores niveles de daño constitutivo al ADN, que se asocia específicamente a la formación de “focis” asociados a Rad52. Las células que carecen Grx5 y Ssq1 son hipersensibles a agentes que dañan el ADN de forma aditiva. Esta sensibilidad es independiente del estado de estrés oxidativo constitutivo que se produce en las células que carecen de Grx5. La ausencia de las proteínas Grx5 y Ssq1 provoca un retraso en la progresión del ciclo celular a través de la fase S. La vía de reparación del ADN, recombinación homóloga podría desempeñar un papel importante en la reparación de los daños en el ADN en las células del mutante Δgrx5.
Glutaredoxins (GRXs) are thiol oxidoreductases widely spread among prokaryotic and eukaryotic organisms. Saccharomyces cerevisiae Grx5 is located at the mitochondrial matrix and participates in the synthesis of iron-sulphur clusters (ISCs), which are co-factors required for several essential cellular processes, such as respiration, regulation of gene expression and DNA-RNA metabolism. Some ISC proteins are involved in DNA metabolism, more precisely in DNA replication and/or repair processes. Recent works reported that defects in ISC metabolism compromise the stability of the cellular genome, and this instability is associated to human predisposition towards multiple types of cancers. Using the yeast grx5 mutant as a model we wanted to gain further insight in the relationship between genomic instability and defects in ISC biosynthesis.The absence of mitochondrial protein Grx5 leads to an increase in genetic instability. This genomic instability is not dependent on the iron accumulation produced in cells lacking Grx5. The cells that do not express Grx5 have higher levels of constitutive DNA damage, specifically associated with the formation of "focis" related with Rad52. Cells lacking Ssq1 and Grx5 are hypersensitive to DNA-damaging agents in an additively way. This sensitivity is independent of the constitutive oxidative stress state that occurs in the cells depleted of Grx5. The absence of proteins Ssq1 and Grx5 causes a delay in cell cycle progression through S phase. The DNA repair pathway homologous recombination could play an important role in the repair of DNA damage in cells Δgrx5 mutant.
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Silva, Almenara de Souza Fonseca. "Influencia de carboidratos na fermentação e na sintese de polissacarideos extracelulares insoluveis pela placa dentaria "in vitro". Ação do ion citrato." [s.n.], 1997. http://repositorio.unicamp.br/jspui/handle/REPOSIP/290492.
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Orientador: Carlos Eduardo PinheiroDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Este trabalho teve como objetivo verificar a influência de carboidratos freqüentemente consumidos na dieta, como glicose, sacarose, frutose e dextrina, na fermentação e na síntese de polis sacarídeos extracelulares insolúveis pela placa dentária in vitro e a inibição destas atividades metabólicas promovida pelo íon citrato. Para tal, foi utilizada uma suspensão de placa dentária (10 mg / ml de tampão fosfato 0,01 pH 7,0), que foi incubada na presença dos substratos a 37°C, durante 2 horas para fermentação e 18 horas para a síntese de polissacarídeos extracelulares insolúveis (PEI). A fermentação foi medida por titulação com solução de NaOH 0,05N. A avaliação da síntese de PEI foi realizada pela dosagem de carboidratos totais. O citrato de sódio foi acrescentado aos meios de incubação, nas concentrações de 50 e 100 mM. Os resultados demonstraram que a glicose produziu a maior quantidade de ácidos e a sacarose sintetizou a maior quantidade de polissacarídeos extracelulares insolúveis, quando omparados com outros carboidratos. O capaz de inibir significativamente a polissacarídeos os extracelulares insolúveis, quando comparados com outros carboidratos. O capaz de inibir significativamente a polissacarídeos os extracelulares insolúveis
Abstract: The purpose of this work was to evaluate the influence of carbohydrates often consumed in diet, as glucose, sucrose, fructose and dextrio, on fermentation and synthesis of extracellular insoluble polysaccarides of dental paque, in vitro and the inhibition of these metabolic activities produced by citrate. So, it was used a suspension of dental paque (10 mg/ml of phosphate buffer 0,01M pH 7,0 ), that was incubated at presence of substrates at 37 'DEGREE' C, for 2 hours for fermentation and 18 hours for synthesis of insoluble, palysaccarides. The fermentation was measured by titration with 0.05N NaOH solution. The estimation of the synthesis of insoluble polysaccarides was achieved by dosage of total carbohydrates. The sodium citrate was increased in an incubation medium on 50 and 100 mM of concentration. The results indicated that the glucose showed the largest acid production activity and sucrose promoted the greatest synthesis of insoluble polysaccarides, when compared with the other carbohydrates. The citrate was able to inhibit the carbohydrate fermentation and showed itself a great inhibitor of synthesis of extracellular insoluble polysaccarides
Mestrado
Fisiologia e Biofisica do Sistema Estomatognatico
Mestre em Odontologia
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Machado, Lisa Santos. "The inhibitory activity of synthetic compounds and ions against transporters of multi-drug resistant bacteria." Master's thesis, Faculdade de Ciências Médicas. Universidade Nova de Lisboa, 2011. http://hdl.handle.net/10362/7712.
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RESUMO: Sessenta e três derivados de hidantoína foram utilizados para avaliar possíveis efeitos de modulação na actividade das bombas de efluxo (BE) na Salmonella NCTC 13349 utilizando um método fluorimétrico semi-automático. Nenhum dos compostos apresentaram actividade anti-bacteriana até concentrações de 240 mg/L. Entre todos os compostos, SZ-7 demonstrou possuir propriedades de modulação de effluxo na presença de glucose. Para testar esta actividade, estirpes de Salmonella resistentes à ciprofloxacina, induzidas a elevados níveis de resistência com sobre-expressão de BE, foram expostas ao SZ-7. Este derivado afectou a susceptibilidade das estirpes à ciprofloxacina.
Uma vez que os 63 compostos estudados apresentaram pouco efeito inibitório /cumulativo, apesar de serem conhecidos pelos seus efeitos moduladores de BE-dependentes de iões em eucariotas, foi questionado o papel dos iões na regulação de BE bacterianas, que poderão influenciar a eficácia de novos compostos. Para este estudo, utilizamos a Escherichia coli AG100 como modelo, devido ao extenso conhecimento no que respeita a estrutura e actividade das BE.
Devido à importância de iões de cálcio (Ca2+) nos canais de transporte membranar e na actividade de ATPases, a sua actividade na modulação do efluxo foi investigada. De resultados anteriormente obtidos concluiu-se que a pH 5 o efluxo é independente de energia metabólica; contudo, a pH 8 é absolutamente dependente, sendo que o Ca2+ é indispensável para manter a actividade das ATPases bacterianas.
A acumulação/effluxo de EtBr pela E. coli AG100 foi determinada na presença/ausência de Ca2+, clorpromazina (inibidor de ligação de Ca2+ a proteínas), e ácido etilenodiamino tetra-acético (quelante de Ca2+). Acumulação/effluxo aumentou a pH 8, contudo o Ca2+ reverte estes efeitos evidenciando a sua importância no funcionamento das BE bacterianas.
Em resumo este trabalho colocou em evidência que muitos aspectos bioquímicos e bioenergéticos devem ser tomados em consideração no estudo da resistência bacteriana mediada por BE.------- ABSTRACT: Sixty-three hydantoin derivatives were evaluated for their modulating effects on efflux pump (EP) activity of Salmonella NCTC 13349 utilizing a semi-automatic fluorometric method. None of the compounds presented antibacterial activities at concentrations as high as 240 mg/L. Among all compounds, SZ-7 showed possible efflux modulating activity in the presence of glucose, indicative of a potential EP inhibitor. To verify its potential effects, ciprofloxacin-resistant Salmonella strains, induced to high level resistance with over-expressing EPs, were exposed to SZ-7. This derivative affected the susceptibility of the ciprofloxacin-resistant strains.
Since the 63 compounds studied had very low inhibitory/accumulative effects, even though their known for being efficient in modulating ion-driven eukaryotic EPs, we questioned whether ions had a leading role in regulating bacterial EPs, influencing the effectiveness of new compounds. For this study we used Escherichia coli AG100 as a model, due to the extensive knowledge on its EPs structure and activity.
Owing the importance of calcium ions (Ca2+) for membrane transport channels and activity of ATPases, the role of Ca2+ was investigated. From previous results we concluded that at pH 5 efflux is independent of metabolic energy; however, at pH 8 it is entirely dependent of metabolic energy and the Ca2+ ions are essential to maintain the activity of bacterial ATPases.
Accumulation and efflux of ethidium bromide (EtBr) by E. coli AG100 was determined in the presence and absence of Ca2+, chlorpromazine (inhibitor of Ca2+-binding to proteins), and ethylenediaminetetraacetic acid (Ca2+ chelator). Accumulation of EtBr increased at pH 8; however Ca2+ reversed these effects providing information as to the importance of this ion in the regulation of bacterial EP systems.
Overall this work puts in evidence that many biochemical and bioenergetic aspects related to the strains physiology need to be taken into consideration in bacterial drug resistance mediated by EPs.
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Osma, Cruz Johann Faccelo. "Production of laccases by the white-rot fungus trametes pubescens for their potential application to synthetic dye treatment." Doctoral thesis, Universitat Rovira i Virgili, 2009. http://hdl.handle.net/10803/8578.
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Approximately 10,000 different dyes and pigments are produced annually worldwide and used extensively in the dye and printing industries. This has resulted in the generation of large volumes of highly polluted wastewater. Apart from the aesthetic deterioration of the natural water bodies, dyes also cause harm to the flora and fauna in the natural environment. Therefore, wastewater containing dyes must be treated prior to their discharge into the environment.Different methods can be applied for the treatment of synthetic dyes from aqueous solutions, such as ozonation, coagulation, flocculation, reverse osmosis and adsorption. However, biological treatments are promising alternatives with different approaches going from the complete immobilization of microorganisms to the pure enzyme utilization. Among all enzymes, laccases are an interesting alternative for the dye degradation due to their low affinity and wide specificity for the substrates. Laccases are multicopper oxidases found in higher plant and microorganisms, like white-rot-fungi; and carry out one-electron oxidation of phenolic and related compounds, and reduce O2 to water.
Thus, this work proposes different strategies based on the use of laccases for the discoloration of synthetic dyes from aqueous solutions. These strategies include studies in different fields to promote eco-friendly solutions for different assets of the whole process. These studies include: the selection of substrates for the production of laccase by the white-rot fungus Trametes pubescens, the possible reutilization of these substrates in the discoloration process, the optimization of the laccase production per culture, the scale up of the laccase production, the use of free and immobilized laccase in the discoloration of dyes and the use of different immobilization techniques to increase the reutilization of the immobilized laccase for the treatment of synthetic dyes.
Aproximadamente 10,000 tintes y pigmentos diferentes son producidos anualmente y tienen un uso extendido en las industrias de teñido e impresión. Esto ha generado grandes cantidades de aguas residuales altamente contaminadas. A parte del deterioro estético que sufren los cuerpos de agua, los tintes también causan daño a la flora y fauna presentes en el medio ambiente. Por ello, las aguas residuales que contienen tintes deben ser tratadas antes de su descarga al ambiente.
Distintos métodos pueden ser empleados para el tratamiento de tintes sintéticos en soluciones acuosas, tales como la ozonización, coagulación, floculación, osmosis inversa y la adsorción. Sin embargo, los tratamientos biológicos resultan una alternativa prometedora con distintas aproximaciones que van desde la inmovilización de microorganismos hasta el uso de las enzimas puras. Entre todas estas enzimas, las lacasas resultan ser muy interesantes para la degradación de tintes debido a su baja afinidad y amplia especifidad por los substratos. Las lacasas son oxidasas de multicobre que se encuentran en plantas, microorganismos, como los hongos de putrefacción-blanca, y llevan a cabo la oxidación del fenol y compuestos relacionados, y reducen el O2 a agua.
Debido a esto, en este trabajo se proponen distintas estrategias basadas en la utilización de las lacasas para la decoloración de tintes sintéticos en medios acuosos. Estas estrategias incluyen estudios en distintas áreas para promover soluciones amigables con el medio ambiente en las distintas etapas del proceso. Estos estudios incluyen: la selección de substratos para la producción de lacasa con el hongo de putrefacción-blanca Trametes pubescens, la posible reutilización de estos substratos en los procesos de decoloración, la optimización de la producción de la lacasa por cultivo, el escalado de la producción de la lacasa, el uso de lacasa libre e inmovilizada en la decoloración de tintes y el uso de distintas técnicas de inmovilización para incrementar la reutilización de la lacasa inmovilizada en el tratamiento de tintes sintéticos.
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Cesarini, Silvia. "Mejora y evaluación de lipasas microbianas para la síntesis de biodiésel = Improvement and evaluation of microbial lipases for biodiesel synthesis." Doctoral thesis, Universitat de Barcelona, 2013. http://hdl.handle.net/10803/129676.
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La presente tesis se ha centrado en la mejora de lipasas de Pseudomonas sp. y en la evaluación de las lipasas microbianas para su aplicación en la síntesis de biodiésel. Diferentes aspectos de este proceso han sido investigados para promover la introducción de la biocatálisis enzimática en la nueva era de la industria, mediante el desarrollo de procesos rentables y sostenibles. Se aplicaron nuevas técnicas de mutagénesis basadas en el diseño racional para mejorar la termoestabilidad de una lipasa psicrófila de Pseudomonas sp. (LipC) con el fin de poderla aplicar en procesos con temperaturas más altas. La variante obtenida (LipCmut) y la proteína salvaje, además de otras lipasas de Pseudomonas sp., fueron eficientemente inmovilizadas en soportes hidrofóbicos. Se puso a punto un protocolo de inmovilización, basado en la adsorción de la enzima a soportes porosos, rápido, de fácil reproducción y de bajo coste, y, por lo tanto, adecuado para un eventual escalado del proceso. Las preparaciones enzimáticas fueron ensayadas en la transesterificación de trioleína como modelo a pequeña escala de síntesis de biodiésel. Cambiando el vector de expresión, mejorando el método de obtención de las lipasas y optimizando las condiciones de transesterificación, se incrementaron las bajas producciones obtenidas inicialmente. Se evaluó el contendido de agua como parámetro fundamental en la síntesis de biodiésel y se introdujo, con éxito, la novedosa alternativa de las lipasas solubles aplicadas en este proceso. Una nueva lipasa soluble desarrollada por Novozymes, Callera Trans L, se ensayó en la transesterificación de aceites crudos, como ejemplo de materia prima más barata. Finalmente, se desarrolló un sistema combinado donde los fosfolípidos (o gomas), presentes en los aceites crudos, son hidrolizados por unas fosfolipasas y los ácidos grasos liberados se esterifican con el metanol para la síntesis final de biodiésel conducida por una lipasa soluble. Esta combinación de desgomado y transesterificación lleva a un proceso de síntesis de biodiésel más barato, más respetuoso con el medio ambiente y, por lo tanto, más sostenible.This thesis is focused on the improvement and evaluation of microbial lipases for biodiesel synthesis. First, the thermal stability of LipC, lipase from Pseudomonas sp. (P. aeruginosa ) was improved. LipC is an enzyme with an interesting psychrophilic behavior and its biochemical characteristics make it a very promising enzyme for biotechnological applications, nevertheless it shows a low thermal stability. Based on the relation structure-function of the protein, a mutagenesis by rational design and saturation of different amino acid positions was performed. A mutant, showing a 7-fold increase in thermal stability at 60°C respect to the wild-type LipC, was obtained. Moreover, this variant kept its cold-adapted properties presenting an optimal activity between 4 and 20°C. Further, immobilization by adsorption of these lipases on different porous supports was carried out. The immobilization is a technique for enzyme stabilization when they have to be applied in industrial biocatalytic processes, such as synthesis of biodiesel. For the immobilization by adsorption, a very fast, efficient, low cost technique, therefore suitable for an eventual process scale, two polypropylene matrices and a silica were tested. Immobilization was performed from the previously obtained mutant (LipCmut) the wild-type (LipC ), its mesophilic homologous (LipA) and another lipase (LipI.3) from Pseudomonas CR611 (P. fluorescens). All lipases were successfully immobilized on the supports tested defining polypropylene Accurel MP1000 as the best carrier. Subsequently immobilized preparations were tested in transformation of triolein into FAMEs (Fatty Acid Metyl Esters) as a small scale model of biodiesel synthesis. LipA, LipCmut and LipC resulted positive for this reaction, but with moderate efficiency. LipCmut and LipC FAMEs production was improved changing their expression vector, increasing the level of protein production and recovery and optimizing the transesterification conditions, in terms of water and methanol content. in this manner, an enormous increase in available lipolytic activity was achieved, corresponding to a higher activity of the immobilized preparations, which were successfully applied for transesterification of soybean oil. The water content in the transesterification reactions was studied in collaboration with Novozymes (Denmark ), where was evaluated a new soluble lipase, Callera Trans L, in the production of biodiesel. Water was found to be an essential factor in the transesterification reaction and a particular reaction mechanism for this lipase, depending on the water and on the free fatty acids, was described. The possibility to apply soluble lipases in a process, so far been carried out only with immobilized enzymes, was an innovation tested also for LipC and LipCmut. The growth culture supernatant, where these extracellular lipases are secreted, was prepared and characterized for the main properties necessary for a transesterification reaction (temperature and methanol resistance). LipC and LipCmut in soluble form resulted to be able to form of FAMEs starting from soybean oil. Moreover, soluble Trans Callera L was tested in the transesterification of crude soybean oil, a substrate difficult for this reaction due to its high content of impurities (free fatty acids and phospholipids). Callera Trans L, showed to be extremely efficient also with this raw material. This result opened the way for the next research which aimed to combine the transesterification with an enzymatic degumming process to remove phospholipids from crude soybean oil. The degumming was carried out with phospholipases that hydrolize phospholipids present in the raw material and prepare it for an efficient transesterification carried out by the lipase. The two processes were performed in the same step, which means an increased sustainability and a substantial reduction in the costs of biodiesel production process.
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Ferre, Malagon Rafael. "Design and synthesis of short antimicrobial peptides for plant protection. Study of their mode of action." Doctoral thesis, Universitat de Girona, 2010. http://hdl.handle.net/10803/8053.
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Aquesta tesi doctoral està basada en el desenvolupament de nous agents antimicrobians derivats del pèptid híbrid cecropina A-melitina WKLFKKILKVL-NH2 (Pep3) que siguin sostenibles i útils per al control de malalties de plantes. Es van dissenyar i sintetitzar més de 133 anàlegs de Pep3 mitjançant química combinatòria. Es van obtenir anàlegs de Pep3 amb una elevada activitat contra fitopatògens i que presentaven baixa toxicitat. Els millors anàlegs van presentar eficàcies comparables amb pesticides de referència en la prevenció d'infeccions causades per fitopatògens. Es va estudiar el mecanisme d'acció de KKLFKKILKYL-NH2 (BP100) investigant la seva interacció amb models de membrana mitjançant tècniques espectroscòpiques. Es va observar la capacitat de BP100 a induir la permeabilització, la neutralització, i l'agregació de vesícules lipídiques aniòniques a una determinada concentració llindar. Es va deduir una equació que relaciona la CMI d'un pèptid antimicrobià amb la constant de partició i la concentració llindar en la membrana.This PhD thesis focused on the development of new sustainable antimicrobial agents derived from the cecropin A-melittin hybrid antimicrobial peptide WKLFKKILKVL-NH2 (Pep3) for use in plant protection. A set of 133 Pep3 analogues were designed and synthetized using a combinatorial chemistry approach. Analogues of Pep 3 with high antimicrobial activity and low toxicity were obtained. The best of them were highly effective preventing infections of phytopathogens being not significantly different to conventional pesticides. Insights into the mode of action of KKLFKKILKYL-NH2 (BP100) were carried out by investigating its interaction with different model membrane systems using spectroscopic methodologies. BP100-induced vesicle permeabilization, membrane electroneutrality, and vesicle aggregation at a defined threshold concentration. Moreover, it was deduced an equation that correlates the MIC of an AMP with the partition constant and the threshold concentration in the membrane.This equation provides an easily prediction of in vivo antimicrobial activities from simple biophysical parameters.
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Vieira, Laura Cardozo. "Obtenção de derivados semissintéticos triterpênicos do ácido ursólico visando atividade biológica." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2013. http://hdl.handle.net/10183/81823.
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As infecções por bactérias representam um grave problema hoje e para o futuro, devido ao fato de que estes microrganismos desenvolvem mecanismos de resistência aos antibióticos ao longo do tempo de uso. A formação de biofilmes também é um fator a ser discutido no cenário atual por estar associado a muitas infecções bacterianas humanas, principalmente àquelas envolvendo dispositivos médicos aumentando assim os riscos de infecções hospitalares. O ácido ursólico (AU) é um triterpeno conhecido por suas atividades biológicas relatadas. Apresenta moderada atividade antibacteriana, porém tem demonstrado importante citotoxicidade frente a algumas linhagens celulares. Em vista disso, neste trabalho se desenvolveu uma série de novas moléculas derivadas do AU com alterações nas posições C-3 e C-28. Quatro moléculas com substituição em C-3 (derivados 2, 3, 4e 5) e uma com substituição em C-3 e C-28 (derivado 6) foram comparadas ao AU (1) quanto a atividade antibacteriana. As cepas utilizadas foram Salmonela Typhimurium, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, Staphylococcus epidermidis e Staphylococcus aureus. Os compostos 3 e 6 apresentaram melhor perfil inibitório de forma geral, onde 3 apresentouse bactericida para S. aureus e S. epidermidis (Gram positivas) e paraP. mirabilis (Gram negativa) apresentou-se bacteriostático.The ursolic acid (UA) is a triterpene known for their biological activities reported. Thus, become useful techniques semi-synthesis starting from natural products extracted, for example residue industries in order to improve the pharmacological properties decreasing toxicity. The bacterial infections are a serious problem today and in the future due to the fact that these organisms develop resistance mechanisms to antibiotics over time of use. The formation of biofilms is also a factor to be discussed in the current scenario because of being responsible for a very high number of rejections and other prosthetic devices by increasing the risk of nosocomial infections. The AU has a moderate antibacterial activity reported in the literature, but showed significant cytotoxicity against some cell lines. In view of this it developed a series of new molecules derived from AU residues extracted from apples (Mallus domestica) from the juice industry by promoting the so-called green chemistry. The molecules undergo changes in C-3 and C-28. Four molecules with substitution at C-3 (derived from 2, 3, 4 and 5) and one with substitution at C-3 and C-28 (derived 6) were compared with au (1). The strains used in the tests of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration were Salmonella Typhimurium, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, Staphylococcus epidermidis and Staphylococcus aureus. Compounds 3 and 6 had better inhibitory profile in general, where three presented bactericidal to S. aureus and S. epidermidis (Gram positive) and P. mirabilis (Gram negative) appeared bacteriostatic.
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Pillay, Nithianandhi. "Microbiological aspects of enterococci isolated at King Edward VIII Hospital, Durban." Thesis, 1999. http://hdl.handle.net/10413/7845.
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The increasing frequency of enterococci as a major cause of nosocomial infections and the transmission of these organisms amongst hospital patients demands a greater awareness of the Enterococcus. Therapy of enterococcal infections is complicated by the pathogens continually changing resistance patterns to many broad-spectrum antibiotics. In addition, the ability of enterococci to cause serious invasive infections including endocarditis and septicaemia with associated high mortality rates; prompted this study which was aimed at identifying the biological properties of enterococci isolated from blood cultures of patients admitted at King Edward VIII hospital, Durban. Enterococci were identified to species level by the API 20 Strep system which identified 68% and a conventional biochemical system of Facklam and Collins which identified 100% of the isolates.The emergence of beta-Iactamase producing enterococci in other countries encouraged the testing of all isolates for this enzyme. All were beta-Iactamase negative. The reported false susceptibility for aminoglycosides and cephalosporins with blood enriched media encouraged the testing of these antibiotics with and without the supplementation of 5% lysed blood. The results showed that an average false susceptibility of 55 % occurred for gentamicin and 35% for tobramycin and netilmicin. The cephalosporins affected, cefotaxime and cefuroxime showed a false susceptibility of 28% and 17% respectively. The choice of treatment for serious enterococcal infections is a syllergistic combination of a beta-Iactam antibiotic plus an aminoglycoside for enterococci with intrinsic low-level resistance. The development of high-level aminoglycoside resistance, MIC 22000,ug/ml results in loss of synergism. This study showed that 26.4 % of enterococcal isolates displayed high level aminoglycoside resistance i.e. to gentamicin and streptomycin. Time-kill study showed reduced killing rate for these organisms for the beta-Iactams and glycopeptides with low-level gentamicin resistance. The results confirmed that a cell-wall active agent combined with gentamicin can be successfully used for enterococcal therapy if the organism has intrinsic low-level resistance to this amino glycoside. Pulsed-field gel electrophoresis (PFGE) carried out on a selected number of Enterococcus faecalis and Enterococcus faecium with high-level aminoglycoside resistance showed a variability in the restriction endonucelase digestion patterns. This suggests independent development of high-level gentamicin resistance and not clonal expression. The ease and reliability with which enterococcal isolates may be typed using this technique to compare different strains represent a significant advance.Thesis (M.Med.Sc.)-University of Natal, 1999.
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Crable, Bryan. "Microbial transformation of 3-nitro-4-hydroxybenzene arsonic acid (roxarsone)." 2007. http://digital.library.duq.edu/u?/etd,108954.
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Kritee, Kritee. "Mass dependent stable isotope fractionation of mercury during its microbial transformations." 2008. http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.000051089.
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Tejasen, Sarun. "Aerobic biotransformation of chlorinated aliphatic hydrocarbons by a benzyl alcohol grown mixed culture : cometabolism, mechanisms, kinetics and modeling." Thesis, 2003. http://hdl.handle.net/1957/30480.
Full textAbstract:
The aerobic transformation of TCE and cis-DCE by a tetrabutoxysilane-grown
microorganism (Vancheeswaran et al., 1999) led to the investigation of novel
substrates, including benzyl alcohol, for promoting cometabolism. The culture grew
on carboxylic compounds and alcohols, but did not grow on formate, methanol,
methane, propane, butane, ethylene, benzene, toluene, or p-xylene. Cis-DCE
transformation was observed when the culture grew on butyrate, glucose, 1-propanol,
1-butanol, ethanol, benzyl alcohol, and phenol, and effectively transformed TCE, cis-DCE, and vinyl chloride when grown on phenol or benzyl alcohol.
Several cycles of growth on benzyl alcohol led to increases in TCE
transformation rates and transformation capacities. Products of benzyl alcohol
degradation shifted from benzaldehyde to 2-hydroxy benzyl alcohol (2HBA) during
the several cycles of growth. In resting cells studies, 2HBA production rates were
highly correlated with TCE transformation rates. TCE transformation and 2HBA
production rates doubled when the culture was grown on phenol and rates of TCE
transformation were correlated with 2HBA production rates. Benzyl alcohol- and
phenol-grown cells oxidized toluene to o-cresol, which indicated the similarity
between benzyl alcohol ortho-monooxygenase, phenol hydroxylase, and toluene
ortho-monooxygenase. 2-Butyne and 1-hexyne (but not acetylene) inhibited benzyl
alcohol- and phenol-grown cells similarly, indicating the same ortho-monooxygenase
was responsible for TCE cometabolism.
Resting cell kinetic studies were performed with cells grown on phenol or
benzyl alcohol. Benzyl alcohol degradation followed a Monod kinetics while phenol
degradation followed a Haldane kinetics. The maximum transformation rates (k[subscript max]) of
TCE, cis-DCE, and VC achieved by phenol-grown cells were about a factor of two
higher than achieved with benzyl alcohol-grown cells, while the half-saturation
constants (K[subscript s]) were in a similar range. Transformation capacities (Tc) for TCE, cis-DCE, and VC were about a factor of two to four higher with phenol-grown cells. The
modeling of TCE, cis-DCE, and VC transformation using independently measured
k[subscript max] and K[subscript s] values matched well with observed data from batch tests. Benzyl alcohol
was shown to be an effective novel substrate for the aerobic cometabolism of TCE,
cis-DCE, and vinyl chloride. Being a non-regulated compound, it might have
applications for in-situ bioremediation.Graduation date: 2004
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Fisher, Edward. "Microbial transformation of arsenic and the characterization of Clostridium sp. strain OhILAs." 2006. http://etd1.library.duq.edu/theses/available/etd-12122006-184030/.
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Hiralal, Lettish. "Effect of fermentation and nutritional conditions on the profile of flavour active ester compounds in beer." Thesis, 2011. http://hdl.handle.net/10413/9061.
Full textAbstract:
During fermentation, the yeast Saccharomyces cerevisiae produces a broad range of aroma-active esters that are important for the desirable complex flavour of beer. The sensory threshold levels of these esters in beer are low, ranging from 0.2 ppm for isoamyl acetate to 15-20 ppm for ethyl acetate. Although esters are only present in trace amounts in beer, they are extremely important as minor changes in their concentration may have dramatic effects on beer flavour. Therefore, optimization of the concentrations of these aroma-active esters in beer is of interest in beer brewing. The number and concentration of esters in beer may be influenced by the fermentation parameters, nutritional composition of fermentation medium and yeast strain type. Therefore, this study investigated the influence of fermentation temperature, pH, and wort nutritional supplements (amino acids and zinc) on the production of yeast-derived ester compounds. In addition, the overall fermentation performance was evaluated based on the reducing sugar and Free Amino Nitrogen (FAN) utilization, ethanol production and yeast cell density. These parameters were analysed using the Dinitrosalicyclic acid method, Ninhydrin assay, Gas Chromatography and standard spread plate technique. The concentration and stability of ethyl acetate, isoamyl acetate, phenyl ethyl acetate, ethyl hexanoate, ethyl decanoate and ethyl octanoate was monitored during storage at 4 °C and room temperature (RT), in the final beer by Chromatography. The expression levels of the ester synthetase genes under conditions that resulted in the highest increase in ester production were quantified by Real-Time PCR. For the lager beer, the best fermentation performance was achieved at RT (±22.5°C), resulting in the utilization of the highest amount of nutrients and production of 4.86% (v/v) ethanol. This was accompanied by the highest production of acetate and ethyl esters, which were 40.86% and 87.21%, respectively, higher than that of the control. Spent yeast density ranged from 2.492 to 3.358 mg/ml for all parameters tested, with the highest yield produced when wort was supplemented with 0.120 g/l zinc sulphate. Fermentations at 14 °C yielded the highest foam head stability and spent yeast viability with a foam head rating of 2.67 and a spent yeast viability of 3.85 × 107 cfu/ml. Ester compounds were relatively stable at 4 °C than at room temperature decreasing by only 7.93% after three months. Of all the volatile esters produced, ethyl decanoate was the least stable, with a 36.77% decrease in concentration at room temperature. For the ale beer, the best fermentation performance which resulted in the highest nutrient utilization was achieved when wort was supplemented with 0.75 g/l L-leucine resulting in the utilization of the highest amount of nutrients (51.25% FAN and 69.11% reducing sugar utilization) and production of 5.12% (v/v) ethanol. At the optimum fermentation pH of 5, 38.27% reducing sugars and 35.28% FAN were utilized, resulting in 4.32% ethanol (v/v) production. Wort supplemented with 0.12 g/l zinc sulphate resulted in 5.01% ethanol (v/v) production and 54.32% reducing sugar utilization. Spent yeast density ranged from 1.985 to 2.848 mg/ml for all parameters tested with the highest yield produced when wort was supplemented with 0.120 g/l zinc sulphate. This was also accompanied by the highest yeast viability of 2.12 × 107 cfu/ml achieved on day 3 of fermentation. Supplementation with 0.75 g/l L-leucine yielded the highest foam head stability with a rating of 2.67. Overall, ester compounds were relatively more stable at 4 °C than at RT decreasing by only 6.93% after three months, compared to a decrease of up to 16.90% observed at RT at the same time. Of all the volatile esters produced, ethyl octanoate was the least stable, with a 32.47% decrease in concentration at RT, phenyl ethyl acetate was the most stable ester at RT, decreasing by 9.82% after three months. Wort supplemented with 0.75 g/l L-leucine resulted in an increase in isoamyl acetate and phenyl ethyl acetate production by 38.69% and 30.40%, respectively, with a corresponding high expression of alcohol acetyltransferases, ATF2 (133.49-fold higher expression than the control). Elevation of fermentation temperature to RT resulted in the upregulation of ATF2 (27.11-fold), and producing a higher concentration of isoamyl acetate. These findings indicate that ester synthesis during fermentation is linked to both substrate availability and the regulation of gene expression. Therefore, it would be possible to manipulate the expression of certain ester synthestase genes to create new yeast strains with desirable ester production characteristics. Results from this study also suggest that supplementing wort with essential nutrients required for yeast growth and optimizing the fermentation conditions could be effective in controlling aroma-active ester concentrations to a desired level in beer.Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.
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Cusimano, Frank Anthony. "Engineered bacteria for the modulation of intestinal physiology, inflammation, and behavior along the microbiome-gut-brain axis." Thesis, 2019. https://doi.org/10.7916/d8-97dx-7887.
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Bacteria in the gastrointestinal tract play an important role in intestinal motility, inflammation, homeostasis, and behavior. Bacteria, through the natural synthesis of neuroactive compounds and secondary metabolites, can modulate the host immune system and communicate with the host along the signaling pathway along the gut-brain axis. Here, we functionally design, develop, test, and characterize a platform for the study of microbial-host interactions using advancements in the field of synthetic biology.
First, we describe the engineering of Escherichia coli Nissle to biosynthesize serotonin within the mammalian gut using a native-plasmid optimized approach. Serotonin is crucial for neurotransmission throughout the body and may be playing a role in microbial gut-brain communication. In the gastrointestinal tract, serotonin regulates intestinal motility, cell turnover, intestinal inflammation, and gastrointestinal homeostasis. Upon serial daily oral gavages, our engineered bacterium populates a murine colon to produce serotonin locally in the mucosa layers along the epithelial lining. Changes in host physiology were observed including decreased gastrointestinal motility, increased colonic Muc2 expression, induction of host TPH2, responsible for serotonin biosynthesis in enteric neurons, and upregulation of serotonin receptors HTR3, HTR4, and HTR7 in the colon. Behavioral tests revealed a statistically significant decrease in anxiety and depression in stress-induced environments in mice treated with the engineered bacterium. This work suggests that gut bacteria engineered to modulate host gut-brain axis may have both scientific and clinical uses to study microbial-host interactions and treat gastrointestinal and behavioral mood disorders in humans.
Second, we engineered bacteria to produce exogenous butyrate and other SCFAs in the murine gut. Short chain fatty acids (SCFAs) play an important role in intestinal homeostasis, fluid dynamics, inflammation, oxidative stress, and intestinal hypersensitivity and motility. With this development, we characterized the effects of our butyrate-producing bacteria on a high-fat diet and DSS-induced colitis model within the colon. Although energetically burdensome to produce, our strains produced butyrate in the colon at higher density in an actively inflamed colitis model. After 14 days of oral administration, our engineered strain (EcN:B) increased the colon length of normal wild-type mice, in high fat fed mice, and in mice with recovering and actively inflamed DSS-induced colitis. EcN:B increased mucosal barrier thickness, upregulated gene expression of the barrier integrity markers Cldn1, Ocln, Zo1, and altered crypt and villus height during inflammation recovery. Furthermore, as butyrate is known to induce Foxp3+ Regulatory T cells, we saw a 13.01% percent increase in Foxp3+ cells in the colon of mice fed our engineered bacteria. This work suggests that synthetic gut bacteria engineered to produce short chain fatty acids may have future clinical uses to treat patients with inflammatory bowel disease including Crohn’s and Colitis with future potential to serve as a therapeutic for irritable bowel syndrome, idiopathic constipation, obesity, and colorectal cancer.
This platform, with the use of synthetic biology to natively engineer Escherichia coli Nissle to produce bioactive compounds in the distal gastrointestinal tract, creates a framework for future characterization of bacterial-host communication and future microbial-based therapeutics.
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Empadinhas, Nuno Miguel da Silva. "Pathways for the synthesis of mannosylglycerate in prokaryotes: genes, enzymes and evolutionary implications." Doctoral thesis, 2005. http://hdl.handle.net/10316/1996.
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Tese de doutoramento em Bioquímica (Microbiologia) apresentada à Fac. de Ciências e Tecnologia de CoimbraOs microorganismos acumulam solutos compatíveis para ajustar a pressão intracelular, protegendo o metabolismo e viabilizando a sobrevivência do organismo em ambientes onde a salinidade ou a secura aumentaram. O manosilglicerato (MG) é um composto frequentemente encontrado em microorganismos de ambientes de elevadas temperatura, como nascentes termais marinhas, sugerindo uma função específica na sua adaptação a altas temperaturas. Esta dissertação caracteriza ao nível bioquímico e molecular as vias metabólicas para a síntese de MG. Na bactéria termófila Rhodothermus marinus foram identificadas duas vias para MG a partir de GDP-manose. Glicerato era o aceitador na via dependente da enzima manosilglicerato sintase (MgS) enquanto que 3-fosfoglicerato era utilizado pela enzima manosil-3-fosfoglicerato sintase (MpgS) para a formação de um intermediário fosforilado que era convertido em MG por uma manosil-3-fosfoglicerato fosfatase (MpgP). Esta via de passo-duplo foi também encontrada no arqueão hipertermófilo Pyrococcus horikoshii e na bactéria termófila Thermus thermophilus. A via de passo-único dependente de MgS era até à data, exclusiva de R. marinus. Os genes envolvidos nestas vias metabólicas foram identificados e clonados, as enzimas correspondentes produzidas em Escherichia coli e caracterizadas bioquimicamente. Os genes mpgS e mpgP eram normalmente adjacentes e em P. horikoshii eram parte dum operão envolvido no metabolismo de manose. A dissertação descreve ainda e caracteriza pela primeira vez um gene e uma enzima para a síntese de MG (mpgS/mpgP) numa bactéria mesófila, Dehalococcoides ethenogenes. Esta novidade evolutiva foi explicada por transferência lateral de genes a partir de arqueões. A recente descoberta de outros genes para MG em procariotas mesófilos e em alguns eucariotas pode reflectir uma pressão selectiva comum independente da adaptação microbiana a elevadas temperaturas. No entanto, a ausência de MG entre os organismos actualmente conhecidos pode indicar perda dos genes resultante duma evolução global da Vida para nichos mais temperados. Ainda assim, a distribuição filogenética dos genes responsáveis pela biossíntese de MG sugere fortemente que esta característica tem uma origem ancestral.
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Silva, Zélia Maria Cordeiro da. "Biological significance of Trehalose for Termus SPP : biochemical characterization of Trehalose synthesis and transport mechanisms in Thermus termophilus." Doctoral thesis, 2006. http://hdl.handle.net/10316/1992.
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Figueiredo, Joana Silva. "Design of strategies to prevent synthesis of S. pneumoniae capsular polysaccharide at the bacteria division septum." Master's thesis, 2012. http://hdl.handle.net/10451/10499.
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Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2012Streptococcus pneumoniae is a common respiratory bacterial pathogen and a frequent cause of community-acquired pneumonia in developed countries. The genes encoding for the capsule polysaccharide (CPS), one of the most important virulence factors of these bacteria, are organized in an operon and in almost all the serotypes the two conserved Wzd and Wze proteins are expressed. Previous results suggest that if these two proteins cannot interact, forming a Wzd/Wze protein complex, pneumococcal bacteria will be prevented from producing capsule at the septum, which was shown to abolish the ability of these bacteria to cause bacteremia in mice after intranasal challenge. In this work, we aimed to find a method capable of screening and identifying small inhibitory (SI) peptides that prevent the interaction between Wzd and Wze. This could, consequently, represent a breakthrough in the development of strategies to replace vaccines against this important clinical pathogen. Initially, a derivative of the Escherichia coli bacterial two-hybrid assay was used. Here, T25- and T18-tagged proteins Wzd and Wze were expressed in the presence of a protein that should compete and interfere with their interaction. However, expression of this control protein, untagged and fully functional Wze, did not prevent the interaction between T25-Wzd and T18-Wze when expressed in a different plasmid. Afterwards we decided to screen for SI peptides directly in S. pneumoniae. For that purpose, we constructed a mutant strain that encodes in the chromosome both proteins, Wzd and Wze, functional and fused to different fluorescent proteins. Accordingly, we observed that Wzd and Wze were localized at the division septum of bacteria and that this localization was lost when a competitor was expressed from a replicative plasmid. We will now screen for SI peptides that can cause delocalization of Wzd and/or Wze and determine their effect on the synthesis of pneumococcal CPS.
Streptococcus pneumoniae é um agente bacteriano patogénico que causa frequentemente pneumonia em paises desenvolvidos. Um dos factores de virulência mais importantes é a cápsula (CPS), um polissacarido que reveste as bactérias. Os genes que codificam para a síntese da cápsula encontram-se organizados num operão que contém os genes wzd e wze, conservados em quase todos os serotipos conhecidos. As proteínas Wzd e Wze interagem formando um complexo proteico que é recrutado para o septo, local de divisão da bactéria, induzindo e regulando a síntese da cápsula. Resultados anteriores sugerem que a inibição da ligação entre estas duas proteinas pode impedir a produção de CPS no septo. Por esta razão, a descoberta de pequenos péptidos, denominados de péptidos SI (pequenos péptidos inibitórios – small inibitory peptides), que inibam a interacção entre as proteínas Wzd e Wze, pode significar uma revolução na criação de estratégias alternativas para substituir as vacinas desenvolvidas contra este patogéneo. O objectivo deste trabalho consistiu no desenvolvimento de um método de identificação de péptidos SI. Começou-se por usar um derivado do sistema “bactérial twohybrid”, em Escherichia coli, em que ambas as proteínas Wzd e Wze, contendo os tags T25 e T18 respectivamente, são expressas na presença de uma proteína competidora capaz de inibir a interacção Wzd/Wze. Contudo, este método não se revelou o mais adequado. De seguida decidiu-se desenvolver um sistema alternativo que pudesse identificar péptidos SI directamente em pneumococos. Para isso, construiram-se mutantes que expressam no cromossoma os genes wzd e wze em fusão com sequências que codificam para diferentes proteínas fluorescentes CFP e Citrine, respectivamente. Esta ferramenta revelou-se capaz de identificar péptidos SI que inibam a interacção entre as proteínas Wzd e Wze. Posteriormente procuraremos outros péptidos SI capazes de deslocalizar as proteínas Wzd e/ou Wze e determinar o seu efeito na síntese de CPS de pneumococos.
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Rodrigues, Tatiana Justo Machado 1986. "Contribution of the Wze protein to the spatial and temporal regulation of capsule synthesis in Streptococcus pneumoniae." Master's thesis, 2010. http://hdl.handle.net/10451/3066.
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Tese de mestrado. Biologia (Microbiologia Aplicada). Universidade de Lisboa, Faculdade de Ciências, 2010As proteínas necessárias à síntese dos polissacáridos capsulares (CPS) de S. pneumoniae estão codificadas num único locus cromossomal, cps, que em quase todos os serótipos tem 4 genes conservados: wzg, wzh, wzd and wze. O terceiro e quarto genes (wzd e wze) codificam as proteínas Wzd e Wze que são homólogas a Wzc, uma proteína de E. coli que integra a família das cinases de tirosinas. Esta família caracteriza-se por ter um dominio transmembranar e um domínio catalítico intracelular que contém os motivos “Walker” A e B, aos quais se liga ATP. Em Proteobactérias estes domínios integram um único polipéptido, mas em bactérias Gram-positivas ambos estão presentes em duas proteínas diferentes: Wzd (proteína transmembranar) e Wze (citoplasmática). A interacção Wzd-Wze é essencial para localização do complexo no septo celular, e pode também ser necessária para modular a síntese de CPS. O objectivo desta dissertação centra-se no estudo da regulação da síntese da cápsula em S. pneumoniae. Especificamente, reproduzimos a estrutura das cinases de tirosinas característica de bactérias Gram-negativas através da ligação covalente entre as proteínas Wzd e Wze. Ao construirmos estas proteínas quiméricas estamos a evitar a necessidade do encontro entre ambas, permitindo-nos investigar o papel do motivo de ligação ao ATP no processo de localização de Wzd-Wze no septo celular. Para tal, examinámos a localização celular das proteínas quiméricas funcionais em S. pneumoniae, in vivo, que mostra uma localização no septo celular. Mostrou-se também que o motivo “Walker” A existente na cinase de tirosinas Wze, é necessário não só para a interacção com Wzd como também desempenha um papel na sua subsequente localização no septo celular. Propomos um modelo no qual cinases de tirosinas típicas de bactérias Gram-positivas possam regular a síntese de CPS através da ligação de ATP e interacção directa/indicrecta com outros componentes da maquinaria de síntese.
The synthesis of the capsular polysaccharides (CPS) in Streptococcus pneumoniae requires several proteins encoded in a single chromosomal locus. Characterization of this capsular polysacharide biosynthesis locus (cps) identifies 4 conserved genes among most serotypes: wzg, wzh, wzd and wze. Wzd and Wze are homologous to Wzc, a member of the bacterial tyrosine-kinase family (BY-kinases), that has a transmembrane domain and an intracellular catalytic domain with canonical Walker A and B motifs that bind to ATP. Both domains exist within a single polypeptide (as Wzc) in Gram-negative bacteria. However, in Gram-positive bacteria those domains are present in two different proteins - Wzd (transmembrane protein) and Wze (cytoplasmic). These proteins interact with each other and this interaction is essential for their localization at the division septum, which is probably required to modulate CPS synthesis. The aim of this thesis was to study the regulation of the capsular polysaccharide synthesis in Streptococcus pneumoniae. Specifically, we reproduced the Gram-negative organization of BY-kinases through the covalent linkage of Wzd to Wze. By generating a Wzd-Wze fusion, we bypass the need for the interaction between the proteins. We then investigated the role of a functional Walker A motif, and ATP binding, in the process of Wzd and Wze localization to the division septum. We examined the cellular localization, in dividing S. pneumoniae, of functional fluorescent derivatives of Wzd-Wze fusions. These chimeric proteins were able to successfully localize to the division septum of unencapsulated bacteria. Moreover, the ATP-binding site of the autophosphorylating protein-kinase Wze, was shown to be required not only for the interaction between Wze and Wzd, but also for septal localization of the complex. We propose a model where Gram-positive BY-kinases may regulate the capsule synthesis through binding of ATP and direct/ indirect interaction with other components of the synthetic machinery.