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Towards the development of selective hydrocarbon oxygenation catalystsGuisado Barrios, Gregorio January 2010 (has links)
The synthesis of pure tris(6-hydroxymethyl-2-pyridylmethyl)amine (H₃L₁₁) is reported for the first time. New complexes of H₃L₁₁ with copper(II), manganese(II) and iron(III) have been characterised by X-ray crystallography. Linear [Fe₃(L₁₁)₂](ClO₄)₃ reveals the tightest Fe-O-Fe angle (87.6°) and shortest Fe...Fe distance (2.834 Å) presently found for a weakly antiferromagnetically-coupled high spin alkoxide-bridged polyiron(III) system. H₃L₁₁ provides a route to various hydrophobic peralkylated TPA ligand derivatives for creating a hydrophobic pocket for the assembly of iron catalysts for the novel 1-hydroxylation of n-alkanes. New 6-py substituted TPA ligands containing methyl (L₁₅) and n-octyl (L₁₆) ether linkages were synthesised via alkylation. Two further novel 6-py substituted ligands were synthesized incorporating n-hexyl substituents on one (L₂₁) and two (L₂₂) of the py moieties. Here a urea spacer group was used to promote hydrogen–bond assisted heterolytic O-O cleavage (generation of the potent FeV=O oxidant) within the hydroxoperoxoiron(III) precursor. High spin [FeII(L)(CH₃CN)[subscript(x)]](CF₃SO₃)₂ complexes (x = 0–2, L = L₁₅,₁₆,₂₁,₂₂) were characterised in solution by ¹H NMR. The structure of [Fe(L₂₂)](CF₃SO₃)₂ reveals a distorted iron(II) centre bound to four N atoms and two urea carbonyls. Iron(II) complexes of H₃L₁₁, L₁₅,₁₆,₂₁,₂₂ and tris(6-Br)-TPA (L₂₄), were investigated for catalysis of the oxygenation of cyclohexane by H₂O₂. Reaction of the iron(II) complexes with H₂O₂ and [superscript(t)]BuOOH was followed by time-resolved EPR and UV-VIS spectrophotometry. A correlation between the observed catalytic activity and the nature of the FeIII(L)-OOR intermediates generated is apparent. A convenient ‘one-pot’ synthesis of benzene-1,3,5-triamido-tris(l-histidine methyl ester) is reported along with attempts at preparing N,N’-bis(pyridylmethyl)-1,3- diaminopropane-2-carboxylic acid (L₂₅), a new water soluble pyridine-amine ligand. The final demetallation step resulted in ligand hydrolysis to the novel amino acid; 1,3-diaminopropane- 2-carboxylic acid which was characterised as its HCl salt by X-ray crystallography.
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Effects of fusion tags on protein partitioning In aqueous two-phase systems and use in primary protein recoveryHassinen, Cynthia January 2002 (has links)
<p>The two techniques aqueoustwo-phase partitioning and expanded bed adsorption that bothare suitable for primary protein recovery were studied. Most ofthe work was focused on partition in aqueous two-phase systemsand in particular on the possibility to effect the partitionbehaviour by fusion of short peptide tags or protein domains tothe target protein.</p><p>The partitioning of fusionproteins between different variants of the domain tag Z and thenaturally occurring protein DNA Klenow polymerase were studiedin Breox/Reppal aqueous two-phase systems. Most studies wereperformed with cell homogenate. The Breox/Reppal system was infocus because if the fusion protein can be partitioned to theBreox-rich top phase the next step can be a thermoseparatingaqueous two-phase system. When the Breox phase is heated to50°C it switches from a one-phase system to a two-phasesystem resulting in an almost pure water rich top phase andhighly concentrated Breox-rich bottom phase. The Breox can thenbe reused and the protein recovered from the water phase. TheZ-domain was genetically modified in different ways to Z<sub>basic1</sub>, Z<sub>acid2</sub>and Z<sub>trp12</sub>and fused to the Klenow protein to try toenhance partitioning to the Breox-rich phase. From theexperiments it was not possible to observe any effects on thepartition behaviour irrespectively of tested properties of thedomain tag. Despite the absence of domain tag effects highK-values, i.e. partition to the Breox-rich top phase, wereobserved in the Breox/Reppal system. However, the proteinK-values seemed to be rather sensitive to the cell homogenateload and showed a tendency to decrease with increased cellhomogenate load. Also increased phosphate concentration reducedthe K-values. The partitioning of cell debris also seemed todependent on the cell homogenate load. At higher homogenateload (<=20g DW/L) clear Breox-rich top phases were observedwith the cell debris collected in Reppal-rich bottomphases.</p><p>Two different tetrapeptides,AlaTrpTrpPro and AlaIleIlePro were inserted near the C-terminusof the protein ZZT0. The Trp-rich peptide unit stronglyincreased both the partitioning of ZZT0 into the poly(ethyleneglycol) (PEG)-rich phase in a PEG/potassium phosphate aqueoustwo-phase system and its retention on PEG and propylhydrophobic interaction chromatographic columns with potassiumphosphate as eluent in isocratic systems. Both the partitioningand the retention increased with increasing number of Trp-richpeptide units inserted into ZZT0. Insertion of Ile-richtetrapeptide units affected the partitioning and retention to amuch lesser extent. Partition and modelling data also indicateda folding of inserted Trp and Ile tetrapeptide units, probablyto minimise their water contact. It was also investigated howto predict the partitioning of proteins in isoelectricPEG/phosphate aqueous two-phase systems.</p><p>The capture ofß-galactosidase from<i>E. coli</i>cell homogentate (50g DW/L) by metal chelatexpanded bed adsorption was studied. These experiments showedthat capture, with a certain degree of selectivity, andclarification of ß-galactosidase could be achieved from acell homogenate. However, a rather low recovery of about 35 %was obtained at a capacity of 0.25mg/mL of gel. Thus, severalparameters remain to be optimised like the load buffercomposition and the cell homogenate load.</p><p><b>Keywords:</b><i>E. coli</i>, aqueous two-phase systems, fusion proteins,hydrophobic interaction chromatography, expanded bedadsorption, ß-galactosidase, Klenow polymerase, Z-domain,peptide tags</p>
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Adsorption of Alkaline Copper Quat Components in Wood-mechanisms and Influencing FactorsLee, Myung Jae 31 August 2011 (has links)
Mechanisms of adsorption of alkaline copper quat (ACQ) components in wood were investigated with emphasis on: copper chemisorption, copper physisorption, and quat adsorption. Various factors were investigated that could affect the adsorption of individual ACQ components in red pine wood. Copper chemisorption in wood was affected by ligand types coordinating with Cu and the stability of the Cu-ligand complexes in solution. For Cu-monoethanolamine (Cu-Mea) system, the prevailing active solvent species at the solution pH, [Cu(Mea)2-H]+ complexes with wood acid sites and loses one Mea molecule through a ligand exchange reaction. The amount of adsorbed Cu was closely related to the cation exchange capacity of wood. An increase in Mea/Cu ratio increased the proportion of the uncharged Cu-Mea complex and resulted in decreased Cu chemisorption in wood. Copper precipitation is also an important Cu fixation mechanisms of Cu-amine treated wood. X-ray diffraction analysis revealed that in vitro precipitated Cu was a mixture of copper carbonates (azurite and malachite) and possibly Cu2O. Higher concentration Cu-amine solutions retarded the Cu precipitation to a lower pH because of higher free amine in the preservative-wood system. The changes in zeta potential of wood in relationship to the quaternary ammonium (alkyldimethylbenzylammonium chloride: ADBAC) adsorption isotherm showed two different adsorption mechanisms for quat in wood: ion exchange reaction at low concentration and additional aggregation form of adsorption by hydrophobic interaction at high concentration. Because of the aggregation effect, when wood was treated with ACQ, high amounts of ADBAC were concentrated near the surface creating a steep gradient with depth. This aggregated ADBAC was easily leached out while the ion exchanged ADBAC had high leaching resistance. Free Mea and Cu of ACQ components appeared to compete with ADBAC for the same bonding sites in wood.
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Persistent organic pollutants (POPs) associated with a platinum mine in the Limpopo Province, South Africa / Ilse JordaanJordaan, Ilse January 2005 (has links)
South Africa ratified the Stockholm Convention (SC), which became legally binding on 17
May 2004. This Convention targets 12 particularly toxic persistent organic pollutants (POPs)
for virtual elimination. The Convention also requires parties to reduce the release of
organochlorine pesticides and the intentionally- and unintentionally-produced POPs such as
dioxins, furans and polychlorinated biphenyls (PCBs) (referred to as dioxin-like chemicals).
Dioxins are a heterogeneous mixture of chlorinated dibenzo-p-dioxins and dibenzofurans
(PCDD/Fs) congeners. These substances were never intentionally produced but are produced
as by-products of industrial processes (such as metallurgical processes and bleaching of paper
pulp). They can also be formed during natural processes such as volcanic eruptions and forest
fires. The largest contributor to releases of PCDD/Fs in the environment is incomplete
combustion from waste incinerators leading to the unintentional production of these
compounds. Polychlorinated biphenyls (PCBs) are used in transformers and capacitors, but
can also be formed unintentionally during industrial and thermal processes. Dioxin-like
chemicals (PCDD/Fs and/or PCBs) are classified as persistent because of the following
characteristics: lipophilicity and hydrophobicity; resistance to photolytic, chemical and
biological degradation and they are able to travel long distances. As South Africa is a semiarid
region, POPs will be less prone to travel here because these substances favour colder
regions with high soil organic matter.
Fish, predatory birds, mammals (including humans) absorb high concentrations of POPs
through the process of bio-concentration, leading to bio-accumulation of these substances in
the fatty tissue. PCDD/Fs occur as unwanted trace contaminants in air, water, land, in
residues and products (such as consumer goods e.g. paper and textiles). The distribution of
these chemicals into various matrices is problematic since they cause damage to the
environment and human health. These chemicals pose a threat to human health when found
in high concentrations that may lead to acute hepatoxicity and dermal toxicity (chloracne).
Long-term exposure to low concentrations of these substances might lead to chronic effects
such as reproductive problems and carcinogenicity.
Since ferrous and non-ferrous metal production is a source of dioxin-like chemicals, a
platinum mine in the Limpopo Province, South Africa, was selected for this investigation.
The aim of the study was to determine if there are dioxin-like chemicals associated with
platinum mining and processing, and if the H4IIE reporter gene bio-assay could be used to
semi-quantify and assess the potencies of the complex environmental and process samples by
determining their Toxic Equivalency Quotients (TEQ). The implications of the sources to the
formation of dioxin-like chemicals regarding the SC were investigated and recommendations
were made to improve this study.
Samples were collected from tailings dams, woodchips, a dumpsite and slag from the smelter
at Union Section. Samples were extracted with the Soxhlet apparatus using hexane as
solvent. The percentage total organic carbon (%TOC) was determined for each sample to
normalise the data. The method used was the Walkley-Black method.
In determining the TEQ of each sample, the H4IIE luc cell line was used. The cells of the
H4IIE luc line are genetically modified rat hepatoma cells stably transfected with a luciferase
firefly gene. The luciferase gene is activated by the presence of dioxin-like compounds and
the concentration of the enzyme is measured as relative light units (RLUs). The amount of
RLUs is directly proportional to the dioxin load in the extract. This method is rapid, cost and
time-effective in determining the TEQ when compared to chemical analysis.
The TEQ2o-valuesin the various samples, as determined with the H4IIE luc cell line, ranged
from 0.007 ngTEQ/kg to 54.06 ngTEQ/kg. Thermal processes at the smelter, sorption of
hydrophobic organic compounds (HOCs) to soil and tailings, and external sources such as
anthropogenic activities contributed to high TEQ2o-values. Climatic conditions, wind,
precipitation, and solubility of HOCs into surfactants lead to low TEQ20. The smelter at
Union Section had a very high TEQ20of 44.62 ngTEQ/kg compared to Impala Platinum mine
(5.15 ngTEQ/kg). This implies that workers at Union Section are possibly exposed to low
and high concentrations of dioxin-like chemicals. Long-term exposure to these compounds
could lead to bio-accumulation in the fatty tissue of the mine workers, leading to chronic
effects such as reproductive problems and cancer. The air emission of the furnace at the
smelter was 0.03 gTEQ/annum and the release of the PCDD/Fs into the slag was 0.60
gTEQ/annum. By effectively managing the smelter it is possible to reduce the TEQ.
The TEQ of each sample increased due to normalising the data. The normalised TEQ20
ranged from 0.94 ng TEQ/kg to 42497.48 ngTEQ/kg.
Dioxin-like chemicals are present on a platinum mine, but at varying quantities and the effects
of these compounds might be detrimental to the environment and the workers at the platinum
mine. Further analyses of the health impacts associated with the platinum mine are needed.
The H4IIE reporter gene bio-assay could be used to effectively determine the TEQ of each
sample. Although this investigation has identified the formation and presence of dioxin-like
chemicals at certain stages of mining and processing, not all of the processes were
investigated. Some of these processes have the potential to add, and even destroy, these
chemicals, affecting potential human exposure and amounts released to the environment.
This, however, requires further investigation.
The financial assistance of the National Research Foundation (NRF) towards this
research is hereby acknowledged. Opinions expressed and conclusions arrived at, are
those of the author and are not necessarily to be attributed to the NRF. / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2006.
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Auto-assemblage supramoléculaire de canaux ioniques vers des matériaux membranaires et des capteurs électro-chimiques macro-organisés / Supramolecular self-assembly of ion and electochemical channels toward macro-organised membrane materials and electrochemical sensorsLe Duc, Yann 17 December 2010 (has links)
L'objectif de ces travaux de thèse concerne l'étude de l'apport de la chimie supramoléculaire à différents niveaux de la science membranaire. Lors de la première partie de nos travaux, nous avons étudié les capacités d'auto-organisation dynamique de molécules bolaformes. Des études de caractérisations ont permis de déterminer différentes structures, dont la formation de canaux ioniques ou aqueux pour certaines. Les capacités de transport de ces molécules auto-assemblés au sein de bicouches lipidiques a été déterminé. Une autre étude concerne l'utilisation d'un milieu hydrophobe, par des interactions supramoléculaire de type forces de Van der Waals, pour confiner de nouvelles fonctionnalisations dans des matériaux mésoporeux. Différents matériaux, dont les utilisations varient avec les molécules confinées, ont été obtenus et caractérisés par plusieurs méthodes d'analyses. Suite à ces résultats, nous avons spécifiquement étudié et optimisé l'utilisation de matériaux silicés électrodéposés sur la surface d'électrodes, puis fonctionnalisés par des chaînes alkyles, pour former des capteurs électrochimiques en étudiant le cas du fullerène. / The main objective of this study concerns the contribution of supramolecular chemistry at different level of membrane science. During the first part of our work, we have studied the dynamic self-organizing capacities of bolaform molecules. Different characterization techniques allowed us to define different structures, including ion or water channels for some of them. Transport capacities of those self-assembling molecules through lipid bilayers have been determined by several tests. Another study is about the use of a hydrophobic environment, by supramolecular interactions such as Van der Waals forces, to confine new functionalization inside mesoporous materials. Different materials, which use is determined by the confined molecules, have been obtained and characterized by several analysis methods. Furthermore, we have studied and optimized the use of electrodeposited silice materials on electrodes surface, then functionalized by alkyl groups, to form electrochemical captors by studying specifically the fullerene case.
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Films polymères minces à base de méthacrylate de glycidyle pour l'élaboration d'interfaces immunoréceptrices : étude par résonance de plasmon de surface / Glycidyl methacrylate based thin polymer films for the elaboration of immunoreceptors interfaces : resonance plasmon surface studyDiop, Dior 17 December 2010 (has links)
Dans ce travail, nous avons cherché à mettre en évidence l'influence de la méthode de préparation de films minces de polymère pour la biofonctionnalisation de surfaces planes. Dans un premier temps, un polymère réactif, le poly(méthacrylate de glycidyle) p(GMA) a été choisi et sa capacité de fixation vis-à-vis d'une biomolécule modèle l'albumine de sérum bovin a été étudiée. Deux stratégies principales de préparation du film polymère ont été utilisées : la technique du grafting onto et celle du grafting from avec deux voies de synthèse : la polymérisation radicalaire classique (PRC) avec l'amorceur en solution et la polymérisation initiée à partir de la surface avec un amorceur photochimique. Il a été montré que la méthode du « grafting from » permettait l'obtention de films d'épaisseur plus élevées que la technique du « grafting onto » avec une meilleure capacité de fixation de biomolécules de BSA. Ces films de p(GMA) se sont révélés relativement hydrophobes, ce qui nous incités à analyser l'influence de la balance hydrophobe/hydrophile des interfaces sur leurs propriétés, dans un second temps. Par la préparation de films copolymères poly(GMA-co-acrylamide) et poly(GMA-co-méthacrylate de glycérol) et la modification des films de poly(GMA) par de l'éthanolamine, l'influence de l'hydrophilie du film sur la capacité de fixation en molécules de BSA et l'activité de reconnaissance moléculaire de celles-ci ont été évaluées. Il a été démontré que par un choix judicieux de la méthode d'hydrophilisation du film polymère, il est possible de réduire considérablement l'adsorption non-spécifique de biomolécules d'où l'obtention de films polymères bioinertes. De plus, les résultats préliminaires ont montré qu'il est possible d'améliorer sensiblement la capacité de reconnaissance moléculaire entre la BSA et son anticorps l'anti-BSA / It is now well accepted that polymeric spacers permit to attach proteins to surfaces efficiently as they carry several binding sites. Moreover, direct attachment of proteins to surfaces might result in the decrease of bioactivity which is critical in the case of the development of biosensors. In this context, we modified gold substrates by polymer grafts via the so-called (i) grafting onto and (ii) grafting from strategies. In (i) preformed polymer chains were attached to the surface, whilst in (ii) surface-confined photopolymerization was performed on either acrylic monomer- or initiator-functionalized gold substrates. The polymer grafts were further biofunctionalized by covalent immobilization of an active protein (bovine serum albumin, BSA). Given the protein-polymer and polymer-gold covalent bonds, the gold/polymer/BSA hybrids permitted to design robust optical biosensors. The modified gold substrates were characterized in terms of chemical composition (X-ray photoelectron spectroscopy), hydrophobicity (contact angle measurements), and polymer coating thickness (surface plasmon resonance, SPR). SPR was also used to monitor in real time the interaction between the grafted antigen to the specific antibody (aBSA). Using unique reactive monomer, glycidyl methacrylate (GMA) in the present case, the implementation of these three methods is assumed to provide polymer films of similar chemical composition but varied interfacial chains conformation. In this respect, influence of polymer chains mobility on the performance of the immunosensing reaction was evaluated. In a further step, hydrophobic/hydrophilic balance of the polymer films was modulated through copolymerization of GMA with acrylamide, and with glycerol methacrylate. It was demonstrated that control over the surface chemical composition of the polymer grafts allows preparing bioinert films, i.e. resistant to non specific adsorption, with enhanced biospecific activity
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Priprema i karakterizacija nanokompozita polimlečne kiseline i silicijum (IV) oksida namenjenog za pakovanje hrane / Preparation and characterization of nanocomposites based on polylactic acid and silica nanoparticles for food packaging applicationRadusin Tanja 13 July 2015 (has links)
<p>Poli(mlečna kiselina) (PLA) predstavlja jedan od najpopularnijih komercijalnih biorazgradivih polimera. Iako može da zameni neke od najčešće korišćenih sintetskih polimera, neka njegova svojstva (loša barijerna, termička i mehanička) još uvek predstavljaju prepreku u široj primeni, posebno za pakovanje hrane. Jedan od najsavremenijih načina prevazilaženja nedostataka u svojstvima biopolimera predstavljaju nanotehnologije.<br />U ovom radu ispitan je uticaj dodatka različitih koncentracija hidrofobnih nanočestica silicijum (IV) oksida (od 0.2 mas.% do 5 mas.%) pripremom uzoraka u rastvoru, i rastopu na toplotna, mehanička, i barijerna svojstva PLA. Morfološke karakteristike uzoraka nanokompozita snimljene su pomoću skenirajuće elektronske mikroskopije (SEM). Ostvarena je izuzetno dobra disperzija i distribucija hidrofobnih čestica silicijum (IV) oksida koje su u malim udelima dodavane u poli(mlečnu kiselinu). Dobra disperzija i distribucija hidrofobnih čestica silicijum (IV) oksida ostvarena je kako pripremom nanokompozita metodom u rastvoru, tako i metodom u rastopu.<br />Toplotna svojstva PLA i pripremljenih nanokompozita proučavana su primenom diferencijalnog skenirajućeg kalorimetra (DSC), dok je stepen kristalnosti određen rasipanjem X zraka pod širokim uglom(WAXD). Mehanička svojstva su ispitivana da bi se odredio uticaj dodatka nanočestica SiO2 na prekidnu čvrstoću i izduženje čistog PLA. Iako su prilikom pripreme materijala metodom u rastvoru, poboljšanja mehaničkih i barijernih svojstava postignuta pri udelima silicijum (IV) oksida u rasponu od 0,2 do 5 mas.%, najznačajnija poboljšanja postignuta su za najmanje udele nanočestica (0,2 mas.% i 0,5 mas.%). Poboljšanja mehaničkih i barijernih svojstava nanokompozita, primenjenih metodom u rastopu, su registrovana i za udele silicijum (IV) oksida od 0,2 do 3 mas.%.<br />Takođe je ispitana mogućnost primene pripremljenog nanokompozita na osnovu poli(mlečne kiseline) i silicijum (IV) oksida za pakovanje prehrambenih proizvoda na primeru pakovanja svežeg svinjskog mesa (M. longissimus thoracis et lumborum). Ispitivanjem uticaja materijala za pakovanje svežeg mesa u vakuumu, na parametre tehnološkog (pH, boja), senzorskog, i mikrobiološkog kvaliteta mesa, utvrđeno je da su PLA kao i nanokompoziti na osnovu PLA sa različitim udelima silicijum (IV) oksida, pogodni za vakuum pakovanje i skladištenje svežeg svinjskog mesa.</p> / <p>Poly(lactic acid) presents one of the most popular bio-polymers for diverse applications. However, the use of PLA as food packaging material is limited due to poor barrier and mechanical properties. These properties could be improved by incorporation of nanoparticles into polymer matrix.<br />In this work neat PLA films and PLA films with different percentage of hydrophobic fumed silica nanoparticles (0,2 wt.% to 5 wt.%) were prepared by solution casting and melt blending methods. Several procedures were used to characterize the influence of different silica content on dispersion (SEM), crystalline behavior (WAXD), thermal stability (DSC, TGA), mechanical and barrier properties. It is shown that the applied techniques and selection of specific hydrophobic spherical nanofiller provide a good dispersion and distribution of silica nanoparticles in poly(lactic acid) for both film preparation methods.<br />Characteristics of films prepared by solution casting method showed improvements in mechanical and barrier properties for all loadings of nanofiller but the most significant improvements were achieved for lowest silica content (0,2 wt.% and 0,5 wt.%) The improvements in material characteristics (mechanical and barrier) for melt blending method were also achieved (for concentrations from 0,2 wt.% to 3 wt.%).<br />After film preparation, selected cuts of M. longissimus thoracis et lumborum were packed in prepred films of polymer nanocomposites, and the shelf-life characterisation was conducted on technological, sensory and microbiological paramethers of quality. After shelf-life characterisation it can be concluded that polymer nanocomposites based on PLA and silica nanoparticles could be used for packaging od fresh pork meat in vacuum.</p>
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Les régulateurs transcriptionnels Rgg. Confirmation de leur implication dans des phénomènes de quorum-sensing et identification de leurs cibles. / RGG transcriptional regulators. Confirmation of their involvement in quorum-sensing phenomenon and identification of their targets.Fleuchot, Betty 06 December 2011 (has links)
La découverte d'un contexte génétique chez les streptocoques – codant un petit peptide hydrophobe (SHP) et un régulateur transcriptionnel appartenant à la famille Rgg –, suivi de l'étude d'un de ces loci chez S. thermophilus LMD-9, a conduit à l'hypothèse que les protéines régulatrices Rgg en association avec une phéromone putative SHP pourraient intervenir dans un mécanisme de type quorum-sensing (QS) chez les bactéries à Gram positif. La première partie de ma thèse a consisté à confirmer cette hypothèse sur le locus shp/rgg1358 de S. thermophilus LMD-9, espèce contenant le plus grand nombre de systèmes SHP/Rgg dans son génome. Pour ceci, les étapes impliquées dans un mécanisme de QS ont été étudiées : la sécrétion, la maturation et la détection à une concentration seuil de la phéromone, sa réimportation à l'intérieur de la cellule, son interaction avec un régulateur transcriptionnel et enfin l'interaction de la protéine régulatrice à l'ADN. Par l'utilisation d'approches génétiques et biochimiques, nous avons démontré l'existence d'un nouveau mécanisme de QS impliquant pour la première fois un régulateur transcriptionnel Rgg et une phéromone SHP, importée à l'intérieur de la cellule par le transporteur d'oligopeptides AmiCDEF. Le rôle de la protéase membranaire, Eep, a également été démontré dans la maturation de la phéromone, dont la forme mature a été déterminée par spectrométrie de masse et validée in vivo. Dans un second temps, nous avons exploré la fonctionnalité de ce nouveau mécanisme sur d'autres loci shp/rgg, dans le but d’étudier l'existence d’éventuels phénomènes de cross-talk entre les bactéries. L'étude de nouveaux loci, en système hétérologue chez S. thermophilus LMD-9, a permis d'étendre la fonctionnalité du mécanisme à deux systèmes SHP/Rgg de streptocoques pathogènes, à savoir S. agalactiae et S. mutans. En parallèle à ce travail de caractérisation, l'identification des régulons des systèmes SHP/Rgg a été entreprise. La construction d'un arbre phylogénétique des protéines Rgg-like a permis d'identifier 68 systèmes SHP/Rgg, que nous avons classés en trois groupes. L'analyse des régions promotrices des gènes shp a conduit à l'identification d'un site putatif de liaison des protéines Rgg à l'ADN spécifiques de chaque groupe SHP/Rgg. Une approche in silico a ensuite été menée afin de rechercher, dans les génomes séquencés de streptocoques, les gènes cibles putatifs. Alors que des cibles proximales ont été détectées pour les groupes II et III, des cibles distales ont été identifiées dans les groupes I et II. Actuellement, la validation de certaines cibles est en cours au laboratoire. A l'avenir, ce travail pourrait permettre le développement de petits peptides permettant d'optimiser l'utilisation de S. thermophilus en industries laitières et de réduire la virulence des streptocoques pathogènes. / The discovery of a genetic context – encoding a small hydrophobic peptide (SHP) and a transcriptional regulator belonging to the Rgg family (in nearly all streptococcal genomes) –, following by the study of one of this loci in S. thermophilus LMD-9, led to the hypothesis that the regulatory proteins Rgg in association with a putative pheromone SHP could define a novel quorum-sensing (QS) regulatory mechanism in Gram-positive bacteria. The first part of my PhD consisted to validate this hypothesis. For this purpose, we analyzed the SHP/Rgg system in all the steps that are commonly involved in QS mechanisms: (i) secretion of the putative pheromone, (ii) maturation of the pheromone, (iii) capture of the pheromone from the external environment at a threshold concentration, (iv) importation of the pheromone inside the cell and (v) interaction of the transcriptional regulator to the promoter regions of targeted genes. Experimentally, we focused on the so-called shp/rgg1358 locus of S. thermophilus LMD-9, which is the streptococcal species containing the largest number of shp/rgg pairs in its genome. By using genetic and biochemistry approaches, we uncovered a new QS mechanism that involves the pheromone SHP, the oligopeptide transporter AmiCDEF for the uptake of the pheromone and the transcriptional regulator Rgg for the control of target gene expression. Furthermore, we showed that the membrane protease Eep participates in the production of the mature pheromone, which has been identified by mass spectrometry. Once characterized, the second part of my PhD was to explore the functionality of this new QS system in other streptococcal strain or species, in order to determine if cross-reactivity phenomenon between streptococci can occur. By using heterologous expression in S. thermophilus LMD-9, we extended the functionality of the SHP/Rgg system to two pathogenic streptococcal species, i.e. S. agalactiae and S. mutans. The last part of my PhD consisted in identifying the regulon of all SHP/Rgg systems. Following the construction of a phylogenetic tree of the Rgg-like proteins in low GC Gram-positive bacteria, we identified 68 SHP/Rgg systems that we classified in three groups. Analyzing the promoter regions of all shp genes led to the identification of a putative Rgg DNA binding site specific to each SHP/Rgg group. An in silico approach was used to scan all sequenced streptococcal genomes for the three identified patterns. Whereas proximal target genes were detected for groups II and III, distal target genes were found in groups I and II. In addition, we uncovered that putative Rgg DNA binding sites can be localized in coding or non-coding region. Currently, validations are in progress. To sum-up, my PhD studies provided evidences that the Rgg proteins in association with small peptide pheromones define a new QS mechanism that seems to regulate the expression of distal and proximal genes in a species-dependent manner. Important insights should be obtained concerning a putative crosstalk among streptococci that involves the SHP/Rgg QS system. My studies may constitute a basis for the development of small peptides to optimize the use of S. thermophilus in dairy factories and reduce the virulence of pathogenic streptococci.
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[en] MULTIOBJETIVE GENETIC ALGORITHM FOR PREDICTING PROTEIN STRUCTURES IN HYDROPHOBIC – POLAR MODEL / [pt] ALGORITMO GENÉTICO MULTIOBJETIVO NA PREDIÇÃO DE ESTRUTURAS PROTEICAS NO MODELO HIDROFÓBICO - POLAREDWIN GERMAN MALDONADO TAVARA 07 October 2014 (has links)
[pt] O problema da predição das estruturas de proteínas (Protein Structure Prediction (PSP)) é um dos desafios mais importantes na biologia molecular. Pelo fato deste problema ser muito difícil, têm sido propostos diferentes modelos simplificados para resolvê-lo. Um dos mais estudados é o modelo, Hidrofóbico-Polar (HP), o modelo HP fornece uma estimativa da energia da proteína com base na soma de interações entre pares de aminoácidos hidrofóbicos (contatos H-H). Entretanto, apesar das simplificações feitas no modelo HP, o problema permanece complexo, pertencendo à classe NP-Difícil. Muitas técnicas têm sido propostas para resolver este problema entre elas, técnicas baseadas em algoritmos genéticos. Em muitos casos, as técnicas baseadas em AG foram usadas com sucesso, mas, no entanto, abordagens utilizando AG muitas vezes não tratam adequadamente as soluções geradas, prejudicando o desempenho da busca. Além disso, mesmo que eles, em alguns casos, consigam atingir o mínimo de energia conhecido para uma conformação, estes modelos não levam em conta a forma da proteína um fator muito importante na hora de obter proteínas mais compactas. Foi desenvolvido um algoritmo genético multiobjetivo para PSP no modelo HP, de modo de avaliar de forma mais eficiente, as conformações produzidas. O modelo utiliza como avaliação uma combinação baseada no número de colisões, número de contatos hidrofóbicos, compactação dos aminoácidos hidrofóbicos e hidrofílicos, obtendo, desta forma estruturas mais naturais e de mínima energia. Os resultados obtidos demonstram a eficiência desse algoritmo na obtenção de estruturas proteicas compactas providenciando indicadores da compactação dos aminoácidos hidrofóbicos e hidrofílicos da proteína. / [en] The problem of protein structured prediction (PSP) is one of the most important challenges in molecular biology. Because this problem is very difficult, different simplified models have been proposed to solve it. One of the most studied is the Hydrophobic-Polar model HP this model provides an estimate of the protein energy based on the sum of hydrophobic contacts. However, despite the simplifications made in the HP model, the problem remains complex, belonging to the class of NP-Hard problems. Many techniques have been proposed to solve this problem as genetic algorithms. In many cases the GA techniques have been used successfully, but, however, with GA approaches often do not adequately address the generated solutions, impairing the performance of the search. Furthermore, in some cases would attain the minimum energy for a known conformation, these models do not take care the protein shape, a very important factor to obtain more compact proteins. This work developed a multiobjective genetic algorithm to PSP in HP model evaluating more efficiently, the conformations produced. This model is a combination of assessment based on the collisions numbers, hydrophobic contacts, hydrophobic and hydrophilic core compression, obtaining thus more natural structures with minimum energy. The results demonstrate the efficiency of this algorithm to obtain protein structures indicators providing compact compression of the hydrophobic and hydrophilic core protein.
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Polymer-Shell Bonded Phase for Improving Online LC-MS Analysis of Intact Proteins, mAbs, and ADCsTse-Hong Chen (7013258) 13 August 2019 (has links)
<p>LC-MS of protein drugs requires new ideas in bonded phase
design rather than adapting bonded phases from the realm of small-molecule
drugs. The polymer-shell bonded phase is designed to interact with larger
molecules and to shield proteins from the silica substrate. The particles
consist of a core of solid silica and a shell of dense polymer brush. The
polymer layer is thick enough to protect the protein from interactions with
silanols to reduce peak tailing. The polymer contains multiple functional
groups that introduce more selectivity. This design gives unprecedented LC
resolution and MS sensitivity. Our group has developed polymer shell bonded
phases for hydrophobic interaction chromatography (HIC-MS) of antibody-drug
conjugates (ADCs), hydrophilic interaction liquid chromatography (HILIC-MS) of
glycoproteins, and reversed-phase liquid chromatography (RPLC-MS) of monoclonal
antibodies. Since HIC is not in-line compatible with MS due to the high salt
levels, it is laborious to identify the constituents of HIC peaks. An
MS-compatible alternative to HIC is reported here: native reversed phase liquid
chromatography (nRPLC). This employs a mobile phase 50 mM ammonium acetate for
high sensitivity in MS, and elution with a gradient of water/isopropanol. The
nRPLC-MS data show that all ADC species, ranging from drug-to-antibody ratios
of 1 to 8, remained intact and native on the column. As we adapt this concept
to intact proteins, we find that lysozyme and α-chymotrypsinogen A are both
eluted in their native conformations. We also use the polymer-shell concept to
resolve IgG1 free thiol variants by RPLC-MS with 0.5% formic acid. Since there
are always other variants besides the intended ones, the need for high MS
sensitivity is desired to distinguish subtle mass change between disulfide bond
and free thiols. Overall, MS sensitivity increases 10X relative while all of
the thiol variants are well resolved by the polymethylmethacrylate bonded phase.</p>
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