Littérature scientifique sur le sujet « PhTAD »

Mycobacterium vanbaalenii PYR-1 is capable of degrading polycyclic aromatic hydrocarbons (PAHs) to ring cleavage metabolites. This study identified and characterized a putative phthalate degradation operon in the M. vanbaalenii PYR-1 genome. A putative regulatory protein (phtR) was encoded divergently with five tandem genes: phthalate dioxygenase large subunit (phtAa), small subunit (phtAb), phthalate dihydrodiol dehydrogenase (phtB), phthalate dioxygenase ferredoxin subunit (phtAc) and phthalate dioxygenase ferredoxin reductase (phtAd). A 6·7 kb EcoRI fragment containing these genes was cloned into Escherichia coli and converted phthalate to 3,4-dihydroxyphthalate. Homologues to the operon region were detected in a number of PAH-degrading Mycobacterium spp. isolated from various geographical locations. The operon differs from those of other Gram-positive bacteria in both the placement and orientation of the regulatory gene. In addition, the M. vanbaalenii PYR-1 pht operon contains no decarboxylase gene and none was identified within a 37 kb region containing the operon. This study is the first report of a phthalate degradation operon in Mycobacterium spp.

Pneumococcal proteins unrelated to serotypes are considered to be candidates of antigens in next-generation vaccines. In the present study, the prevalence of vaccine candidate protein genes, along with serotypes and antimicrobial resistance determinants, was investigated in a total of 57 isolates obtained from a tertiary care hospital in Japan. All of the pediatric isolates and 76.6% of the adult isolates did not belong to PCV13 (a 13-valent pneumococcal conjugate vaccine) serotypes, and 70.2% of all isolates showed multidrug resistance. All of the isolates had ply, pavA, nanA, and nanB, and high prevalence was noted for the pspA and pspC genes (96.5% and 78.9%, respectively). Detection rates for the pneumococcal histidine triad protein (Pht) genes phtA, phtB, phtD, and phtE were 49.1%, 26.3%, 61.4%, and 100%, respectively. Two fusion-type genes, phtA/B and phtA/D, were identified, with a prevalence of 36.9% and 14.0%, respectively. These fusion types showed 78.1–90.0% nucleotide sequence identity with phtA, phtB, and phtD. The most prevalent pht profile was phtA + phtD + phtE (26.3%), followed by phtA/B + phtE (19.3%) and phtA/B + phtD + phtE (17.5%), while pht profiles including phtD and/or phtA/phtD were found in 71.9% of isolates. The present study revealed the presence of two fusion types of Pht and their unexpectedly high prevalence. These fusion types, as well as PhtA and PhtB, contained sequences similar to the B cell epitopes that have been previously reported for PhtD.

ABSTRACT Four pneumococcal genes (phtA, phtB, phtD, andphtE) encoding a novel family of homologous proteins (32 to 87% identity) were identified from the Streptococcus pneumoniae genomic sequence. These open reading frames were selected as potential vaccine candidates based upon their possession of hydrophobic leader sequences which presumably target these proteins to the bacterial cell surface. Analysis of the deduced amino acid sequences of these gene products revealed the presence of a histidine triad motif (HxxHxH), termed Pht (pneumococcal histidine triad) that is conserved and repeated several times in each of the four proteins. The four pht genes (phtA, phtB, phtD, and a truncated version of phtE) were expressed inEscherichia coli. A flow cytometry-based assay confirmed that PhtA, PhtB, PhtD and, to a lesser extent, PhtE were detectable on the surface of intact bacteria. Recombinant PhtA, PhtB, and PhtD elicited protection against certain pneumococcal capsular types in a mouse model of systemic disease. These novel pneumococcal antigens may serve as effective vaccines against the most prevalent pneumococcal serotypes.

Restricted to the genus Streptococcus, the Pht protein family comprises four members: PhtA, PhtB, PhtD and PhtE. This family has the potential to provide a protein candidate for incorporation in pneumococcal vaccines. Based on sequence analysis and on RT-PCR experiments, we show here that the pht genes are organized in tandem but that their expression, except that of phtD, is monocistronic. PhtD, PhtE, PhtB and PhtA are present in 100, 97, 81 and 62 % of the strains, respectively, and, by analysing its sequence conservation across 107 pneumococcal strains, we showed that PhtD displays very little variability. To analyse the physiological function of these proteins, several mutants were constructed. The quadruple Pht-deficient mutant was not able to grow in a poor culture medium, but the addition of Zn2+ or Mn2+ restored its growth capacity. Moreover, the phtD mRNA expression level increased when the culture medium was depleted in zinc. Therefore, we suggest that these proteins are zinc and manganese scavengers, and are able to store these metals and to release them when the bacterium faces an ion-restricted environment. The data also showed that this protein family, and more particularly PhtD, is a promising candidate to be incorporated into pneumococcal vaccines.

ABSTRACTPneumococcal adherence to mucosal surfaces is a critical step in nasopharyngeal colonization, but so far few pneumococcal adhesins involved in the interaction with host cells have been identified. PhtA, PhtB, PhtD, and PhtE are conserved pneumococcal surface proteins that have proven promising as vaccine candidates. One suggested virulence function of Pht proteins is to mediate adherence at the respiratory mucosa. In this study, we assessed the role of Pht proteins in pneumococcal binding to respiratory epithelial cells. Pneumococci were incubated with human nasopharyngeal epithelial cells (Detroit-562) and lung epithelial cells (A549 and NCI-H292), and the proportion of bound bacteria was measured by plating viable counts. Strains R36A (unencapsulated), D39 (serotype 2), 43 (serotype 3), 4-CDC (serotype 4), and 2737 (serotype 19F) with one or more of the four homologous Pht proteins deleted were compared with their wild-type counterparts. Also, the effect of anti-PhtD antibodies on the adherence of strain 2737 to the respiratory epithelial cells was studied. Our results suggest that Pht proteins play a role in pneumococcal adhesion to the respiratory epithelium. We also found that antibody to PhtD is able to inhibit bacterial attachment to the cells, suggesting that antibodies against PhtD present at mucosal surfaces might protect from pneumococcal attachment and subsequent colonization. However, the relative significance of Pht proteins to the ability of pneumococci to bindin vitroto epithelial cells depends on the genetic background and the capsular serotype of the strain.

ABSTRACTStreptococcus pneumoniaeis a major human pathogen responsible for massive global morbidity and mortality. The pneumococcus attaches a variety of proteins to its cell surface, many of which contribute to virulence; one such family are the polyhistidine triad (Pht) proteins PhtA, PhtB, PhtD, and PhtE. In this study, we have examined the mechanism of Pht surface attachment using PhtD as a model. Analysis of deletion and point mutants identified a three-amino-acid region of PhtD (Q27-H28-R29) that is critical for the process. The analogous region in PhtE was also necessary for its attachment to the cell surface. Furthermore, we show that a large proportion of the total amount of each Pht protein is released into bacterial culture supernatants. Other surface proteins were also released, albeit to lesser extents, and this was not due to pneumococcal autolysis. The extent of release of surface proteins was strain dependent and was not affected by the capsule. Lastly, we compared the fitness of wild-type and ΔphtABDEpneumococciin vivoin a mouse coinfection model. Release of Pht proteins by the wild type did not complement the mutant strain, consistent with surface-attached rather than soluble forms of the Pht proteins playing the major role in virulence. The significant degree of release of Pht proteins from intact bacteria may have implications for the use of these proteins in novel vaccines.

Introduction. Streptococcus pneumoniae is a significant bacterial pathogen in humans. Currently, there are two types of pneumococcal vaccines, but there are concerns regarding their application. Aim. Since many pneumococcal proteins are serotype-independent, polyhistidine triad protein D (PhtD) has been selected as a vaccine candidate. Methodology. We prepared recombinant PhtD and its C-terminal fragment (PhtD-C) using alum and outer-membrane vesicles (OMVs) as adjuvants. The combinations were injected intraperitoneally into mice, and then total immunoglobulin G (IgG) and specific IgG, IgG1 and IgG2a were measured. A serum bactericidal assay and opsonophagocytosis were also performed as complementary tests. Meningococcal OMVs were used as an adjuvant. Results. The levels of specific IgG and IgG1 against combinations of PhtD and its C-terminal with OMVs and alum as adjuvants increased at the time of the third mouse immunization on day 35. Forty per cent and 60% of S. pneumoniae ATCC 6303 (serotype 3) as a virulent pneumococcal strain, respectively, were killed in the opsonophagocytosis test and these results could also be observed in the serum bactericidal assay. Mice mmunized iwith PhtD and its C-terminal with OMVs and alum as adjuvants survived after 10 days of pneumococcal challenge. Conclusion. The combination of PhtD and PhtD-C with alum produced optimal results, but the combination of PhtD and PhtD-C with OMVs produced minimal results by comparison. The survival rates were also measured, and these corresponded with the results of the immunological assessments. Our findings showed that mice receiving PhtD and PhtD-C plus OMV and alum had higher survival rates than the mice in the other groups.

I consider Phad Painting as a valuable pilgrimage of Rajasthan. Phad painting (Mewar Style of painting) or Phad is a stye religious scroll painting and folk painting, practiced in Rajasthan state of India. This is a unique scroll making folk art; this style of painting is traditionally done on a long piece of cloth or canvas, known as phad. It is synonymous with the Bhopa community of the state. These are beautiful specimen of the Rajasthani cloth paintings. The narratives of the folk deities of Rajasthan, mostly of Pabuji and Devnarayan- who are worshipped as the incarnation of lord Vishnu & Laxman. Each hero-god has a different performer-priest or Bhopa. The repertoire of the bhopas consists of epics of some of the popular local hero-gods such as Pabuji, Devji, Tejaji, Gogaji, Ramdevji.The Phad also depict the lives of Ramdev Ji, Rama, Krishna, Budhha & Mahaveera. The iconography of these forms has evolved in a distinctive way. Shahpura in Bhilwara district of Rajasthan are widely known as the traditional artists of this folk art-form for the last two centuries. Presently, Shree Lal Joshi, Nand Kishor Joshi, Prakash Joshi and Shanti Lal Joshi are the most noted artists of the phad painting, who are known for their innovations and creativity. Noted examples of this art are Devnarayan Ki Phad and Pabuji Ki Phad.

Una delle principali applicazioni in cui i polimeri elastomerici trovano impiego è come componente strutturale nella formulazione di mescole per pneumatici. Tuttavia, affinché il prodotto finale possa soddisfare gli standard di prestazione richiesti, è necessario introdurre nella matrice polimerica dei componenti (filler) inorganici (Silice e Carbon Black). Sotto questo profilo, la compatibilità tra la fase polimerica e inorganica è dunque un aspetto di cruciale importanza. In questo contesto si colloca il presente progetto di dottorato, che ha come obiettivo quello di sviluppare un’innovativa strategia per migliorare il composito finale attraverso l’introduzione di gruppi funzionali sulla matrice polimerica in grado di interagire con le componenti inorganiche presenti in una mescola. Come prima cosa sono state scandagliate le opportunità che la letteratura offriva per trovare una reattività in grado di interessare i legami insaturi presenti nella matrice polimerica. In particolare, sono state identificate tre possibili alternative:cicloaddizione 1,3-dipolare, reazioni di tipo Tiol-Ene e reazioni di tipo Alder-Ene. Dopo aver verificato pregi e difetti di ciascuna reattività, la più promettente si è rivelata essere quella basata su una reazione di tipo Alder-Ene, che prevede l’interazione tra un’olefina, recante idrogeni in posizione allilica, ed un enofilo elettron-povero. Il composto enofilo da noi utilizzato e studiato come sistema modello è stato il 4-phenyl-1,2,4-triazoline-3,5-dione (PhTAD). Questo sistema, una volta ancorato sul polimero, presenta un gruppo ammidico secondario libero, in grado di modificare localmente la polarità della matrice e, contemporaneamente, di interagire tramite legami ad idrogeno con i filler inorganici come la silice. L’attività di ricerca è stata dunque concentrata sulla modifica chimica, con PhTAD, di olefine commerciali. Tali polimeri modificati, con diverse quantità di funzionalizzante, sono stati quindi caratterizzati tramite un approccio multitecnica (DSC, TGA, FTIR e prove di swelling) ed in seguito introdotti in mescola. Le proprietà reologiche delle mescole ottenute sono state valutate sia con un reometro a disco oscillante (ODR) sia tramite analisi dinamico meccaniche a temperatura variabile (DMTA) ed analizzandone le curve di stress-strain. Si è quindi tentato di risolvere le criticità emerse nell’utilizzo di PhTAD come agente di funzionalizzazione. Innanzitutto, è stato necessario ottimizzarne la quantità introdotta in mescola arrivando, nel migliore dei casi, ad ottenere compositi in cui i valori di G’ fossero comparabili con gli standard di rifermento industriali, che si basano sull’utilizzo di agenti compatibilizzanti come il TESPT, con un contestuale leggero peggioramento dei valori come il tanδ o l’effetto Payne, indice di un’effettiva interazione tra la matrice polimerica funzionalizzata con le cariche di silice, seppur non ottimale. Inoltre, uno dei maggiori problemi intrinseci da risolvere nell’utilizzo di una molecola come il PhTAD, risiede nella sua alta reattività che rende impossibile operare in massa, direttamente sul polimero pristino. L’ultima parte del progetto è stata quindi dedicata alla sintesi di funzionalizzanti di natura simile, a base diazenica, ma meno reattivi, allo scopo di far avvenire la reazione sul polimero direttamente in fase di formulazione, alle temperature a cui vengono processate le mescole (≈140°C), svincolandosi così dalla difficoltà derivanti dell’operare in soluzione. In particolare,l’avvenuta funzionalizzazione di un sistema oligomerico modello con ethyl(anilinocarbonyl)diazenecarboxylate ha permesso di dimostrare la validità dell’idea di una funzionalizzazione in massa termicamente stimolata, aprendo alla possibilità di impiego di altri sistemi molecolari, modificabili appositamente per assolvere una funzione specifica all’interno della mescola.
One of the main applications in which elastomeric polymers are used is as a structural component in the formulation of tire compounds. However, to match the required performance standards for the final product, it is necessary to introduce inorganic components (fillers) (Silica and Carbon Black) into the polymer matrix. From this point of view, the compatibility between the polymeric and inorganic phases is, therefore, an aspect of crucial importance. In this context the present PhD project is set up, which aims to develop an innovative strategy to improve the final composite through the introduction of functional groups on the polymer matrix able to interact with the inorganic components present in a compound. First, the opportunities offered by the literature to find a reactivity able to affect the unsaturated bonds present in the polymer matrix were explored. In particular, three possible alternatives have been identified: 1) 1,3-dipolar cycloaddition 2) Tiol-Ene type reactions 3) Alder-Ene type reactions After having verified the strengths and weaknesses of each reactivity, the most promising one has been to be based on an Alder-Ene reaction, which involves the interaction between an olefin (bearing hydrogen in an allyl position) and an electron-poor enophile. The enophilic compound we studied as a model system was 4-phenyl-1,2,4-triazoline-3,5-dione (PhTAD). This system, once anchored on the polymer, has a secondary amide group, able to modify locally the polarity of the matrix and, at the same time, able to interact through hydrogen bonds with inorganic fillers such as silica. The research activity has been focused on the chemical modification, with PhTAD, of commercial unsaturated polymers. These modified polymers, with different amounts of functionalizer, were then characterized by a multitechnical approach (DSC, TGA, FT-IR and swelling tests) and subsequently introduced into the mixture. The rheological properties of the compounds obtained were evaluated both with an oscillating disk rheometer (ODR), and by dynamic mechanical analysis at variable temperatures (DMTA) and by analyzing stress-strain curves. Preliminary attempts have been performed to resolve the critical issues that emerged when using PhTAD as a functionalization agent. First, it was necessary to optimize the amount introduced into the mixture, arriving, at best, to obtain composites in which the values of the G' module were comparable with the industrial reference standards, which are based on the use of compatibilizing agents such as TESPT, with a slight contextual deterioration of the values such as tanδ or the Payne effect, indicating an effective interaction between the polymer matrix functionalized with silica fillers, even if not yet optimally. Moreover, one of the major intrinsic problems to be solved in the use of a molecule such as PhTAD lies in its high reactivity which makes it impossible to operate in bulk, directly on the pristine polymer. The last part of the project was then dedicated to the synthesis of functionalizers of a similar nature, based on diazenics, but less reactive, in order to make the reaction occur on the polymer directly in the formulation phase, at the temperatures normally used to process compounds (≈140 °C), thus avoiding the difficulty due to operating in solution. The successful functionalization of a model oligomeric system with ethyl (anilinocarbonyl) diazenecarboxylate has allowed to demonstrate the validity of the idea of a thermally stimulated mass functionalization, opening to the possibility of using other molecular systems, that can be tuned specifically to perform a specific function within the compound.

ABSTRACT Aquifer storage and recovery (ASR) is used world-wide to supplement available water supplies by storing surplus water in aquifers and recovering it during periods of drought and increased demand. The use of ASR as an option for increasing available municipal irrigation and fresh water supplies is threatened as a result of the mobilization of arsenic in some aquifers during ASR. Arsenic is liberated from arsenic-bearing sulfide minerals as a result of the mixing of oxidizing injected water with reducing insitu groundwater. Fracture networks can have significant influence on the migration and distribution of arsenic in the Upper Floridan Aquifer (UFA) during ASR operations through effects on fluid flow, chemical reactions, and transport characteristics. To characterize fracture flow and associated mass transport, numerical three-dimensional models constructed with MODFLOW and FracMan are used to represent fractures in equivalent continuum, discontinuum, and stochastic discontinuum or discrete fracture network (DFN) dual porosity or hybrid models. The geochemical reaction (PHREEQC-2) and transport (MT3DMS) models are coupled to the three dimensional numerical flow model (MODFLOW 2000) as PHT3D- 2003, and utilized to simulate the flow, transport, and inorganic reactions associated with the injection of oxidized water into the UFA of Southwest Florida during ASR cycles. The discrete fractures, implicitly simulated in MODFLOW as high flow zones, are model layers of varying thicknesses with uniform hydraulic conductivity and storage parameters, and as stochastically-generated horizontal and vertical fractures with varying physical attributes including orientation, aperture widths, fracture intensity, and fracture distributions, distributed within a lower conductivity matrix. Discrete fracture networks are simulated with FracMan and the results imported into MODFLOW. Although each fracture zone layer is assigned a unique stochastic distribution of hydraulic conductivity, each model represents a single realization. The FracMan output of stochastic distributions of hydraulic conductance and storage parameters is "upscaled" for use in MODFLOW. The vertical migration of solute due to variations in the density of injectate and groundwater does not appear to be a significant characteristic of the modeled flow system. The modeling results support the hypothesis that arsenopyrite, which is stable under reducing conditions, liberates arsenic during recharge cycles as a result of oxidation. The results also indicate that fracture flow significantly controls the distribution of all solutes affected by the ASR flow system due to the significantly higher transmissivity of the fractures compared to the matrix. The simulated distribution of arsenic in the matrix is significantly less than in the fractures as a result of the limited penetration of oxidized recharge waters into the inter-fracture matrix. Under the simulated aquifer and geochemical conditions, arsenic travels farther from the injection well via fractures than is observed in monitor wells, suggesting that the partially-penetrating monitoring well network does not intercept many of the fractures. The modeled increases in concentrations of arsenic in the ASR wells during the recovery cycles are also consistent with observations. Explicit representation of fracture zones in numerical transport models provides an increased understanding of the flow system and the potential occurrence and distribution of arsenic in groundwater.

A grande reatividade da 4-fenil-1,2,4-triazolina-3,5-diona (PhTD) (2) como dienófilo nas reações Diels-Alder e enófilo nas reações Ene, tem sido amplamente estudada na literatura. Entretanto poucos estudos tem sido desenvolvidos para elucidar o mecanismo via substituição eletrofílica aromática de reações com triazolinas de caráter eletrofílico. O polímero Poli(oxi-2,6-dimetil-1,4-fenileno) (PPE) (1) é de importância industrial como componente de plástico de engenharia, devido a sua alta temperatura de transição vítrea (Tg = 205 +- 20 °C), e por exibir completa miscibilidade com o poliestireno. Para se obter materiais com melhor desempenho, várias tentativas tem sido realizadas na modificação química do PPE procurando gerar novos polímeros e copolímeros. Este polímero modificado (3) com grupamentos triazolinadiona poderá apresentar maior interesse industrial através da inserção de grupos fortemente polares capazes de formar pontes de hidrogênio e/ou levar à formação de ionômeros. Apesar da importância que o PPE representa no meio industrial e o que poderá representar quando modificado com grupamentos polares, como triazolinadionas, ainda há uma obscuridade com relação ao mecanismo envolvido na formação do PPE-UZ. Assim neste trabalho um estudo cinético foi desenvolvido usando métodos espectroscópicos (UV-Visível e 1H-RMN), para o monitoramento da reação entre o PPE e PhTD. A partir de soluções de PhTD e PPE em clorobenzeno realizou-se uma série de experimentos da reação entre PhTD e PPE em condições de pseudo-primeira ordem com relação à concentração de PhTD. Foram também realizados experimentos com o trímero do PPE com a finalidade de eliminar as contribuições associadas a cadeia polimérica. As constantes de velocidades e parâmetros de ativação foram comparados entre ambos sistemas. A análise dos resultados experimentais indicam que a reação ocorre em mais de uma etapa e com formação de um complexo de natureza sigma. Sua existência foi evidenciada com o aparecimento de um sinal na região entre 4-5 ppm quando a reação foi monitorada por 1H-RMN, também nos experimentos utilizando substratos competitivos com isopreno e base (4-aminobenzoatoa de etila), a qual acelera a etapa que leva a formação do produto final. A reação mostrou uma dependência linear das constantes de velocidade com a concentração do polímero e com a temperatura. As condições de pseudo-primeira ordem foram mantidas, pois a equação de Arrhenius foi obedecida. Também foi observado o comportamento destas constantes de velocidades com a variação da polaridade do meio reacional.
4-phenil-1,2,4-triazoline-3.4-dione (PhTD) (2) due to its great reactivity as a dienophile in Diels-Alder reactions and enophile in Ene reactions has been extensively studied. However only a few investigations have been reported on the mechanism via eletrophilic aromatic substitution in reactions with eletrophilic triazolines. Poly(oxy-2,6-dimethyl-1,4-phenylene) (PPE) (1) is a polymer of great industrial importance as a component in engineering plastics due to its high glass transition temperature (Tg = 205 +- 20 °C). The modified polymer (3) may exhibit some interesting properties due to the insertion of very polar groups capables of forming hidrogen bridge bonds and undergoing dissociation process, wich could lead to ionomers. Although of importance this polymer play in industrial centre and that could to play when modified with polar groups as triazolinediones still little is known with regard to the mechanism involved in the formation of PPE-UZ. In this work a kinetic study was carried out for the reaction between PPE and PhTD using spectroscopical methods as UV-Visible and 1H-RMN. Starting from solutions of PhTD and PPE in chlorobenzene a series of reactions were carried out under pseudo-first order conditions by keeping constant the concentration of PhTD. In order to evaluate the reaction kinetics a similar study was performed with oligomeric PPE (trimer). Comparison of the kinetic parameter for the reaction between the PPE and PhTD and for the reaction between the oligomeric PPE, allowed considerations to be made on the isolating polymer backbone contributions. Analysis of the experimental results showed that the reaction occurs in more than one step. The initial sleeps led to the formation of a sigma-complex as it was evidenced by addition of a competing substrate, isoprene, and base (ethyl 4-aminobenzoate) which accelerated the step that lead to the final product. This intermediate was also detected by 1H-RMN, though the appearence of signals at 4-5 ppm, characteristic of olefinic proton (arenium ion). The reaction obeyed kinetic pseudo first order conditions. No deviation for the Arrhenius equation was detected. Solvent dieletric constant showed a marked effect on the rate constants.