Structural and functional characterization of Group B Streptococcus class C sortases

In Group B Streptococcus (GBS) three structurally distinct types of pili have been discovered as potential virulence factors and vaccine candidates. The pilus-forming proteins are assembled into high-molecular weight polymers via a transpeptidation mechanism mediated by specific class C sortases. Using a multidisciplinary approach including bioinformatics, structural and biochemical studies and in vivo mutagenesis we performed a broad characterization of GBS sortase C. The high resolution X-ray structure of the enzymes revealed that the active site, located into the β-barrel core of the enzyme, is made of the catalytic triad His157-Cys219-Arg228 and covered by a loop, known as the “lid”. We show that the catalytic triad and the predicted N- and C-terminal trans-membrane regions are required for the enzyme activity. Interestingly, by in vivo complementation mutagenesis studies we found that the deletion of the entire lid loop or mutations in specific lid key residues had no effect on catalytic activity of the enzyme. In addition, kinetic characterizations of recombinant enzymes indicate that the lid mutants can still recognize and cleave the substrate-mimicking peptide at least as well as the wild type protein.

Polymerizing activity and regulation of group B Streptococcus pilus 2a sortase C1

Group B Streptococcus [GBS; Streptococcus agalactiae] is the leading cause of life-threatening diseases in newborn and is also becoming a common cause of invasive diseases in non-pregnant, elderly and immune-compromised adults. Pili, long filamentous fibers protruding from the bacterial surface, have been discovered in GBS, as important virulence factors and vaccine candidates. Gram-positive bacteria build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates. Despite the availability of several crystal structures, pilus-related C sortases remain poorly characterized to date and their mechanisms of transpeptidation and regulation need to be further investigated. The available three-dimensional structures of these enzymes reveal a typical sortase fold except for the presence of a unique feature represented by an N-terminal highly flexible loop, known as the “lid”. This region interacts with the residues composing the catalytic triad and covers the active site, thus maintaining the enzyme in an auto-inhibited state and preventing the accessibility to the substrate. It is believed that enzyme activation may occur only after lid displacement from the catalytic domain. In this work we provide the first direct evidence of the regulatory role of the lid, demonstrating that it is possible to obtain in vitro an efficient polymerization of pilin subunits using an active C sortase lid mutant carrying a single residue mutation in the lid region. Moreover, biochemical analyses of this recombinant mutant reveal that the lid confers thermodynamic and proteolytic stability to the enzyme. A further characterization of this sortase active mutant showed promiscuity in the substrate recognition, as it is able to polymerize different LPXTG-proteins in vitro.

Measurement of the $B^0$ - $\bar{B}^0$ and $B^0_s$ - $\bar{B}^0_s$ production asymmetries in pp collisions at $\sqrt{s}=7$ TeV and $\sqrt{s}=8$ TeV with the LHCb experiment

The production rate of $b$ and $\bar{b}$ hadrons in $pp$ collisions are not expected to be strictly identical, due to imbalance between quarks and anti-quarks in the initial state. This phenomenon can be naively related to the fact that the $\bar{b}$ quark produced in the hard scattering might combine with a $u$ or $d$ valence quark from the colliding protons, whereas the same cannot happen for a $b$ quark. This thesis presents the analysis performed to determine the production asymmetries of $B^0$ and $B^0_s$. The analysis relies on data samples collected by the LHCb detector at the Large Hadron Collider (LHC) during the 2011 and 2012 data takings at two different values of the centre of mass energy $\sqrt{s}=7$ TeV and at $\sqrt{s}=8$ TeV, corresponding respectively to an integrated luminosity of 1 fb$^{-1}$ and of 2 fb$^{-1}$. The production asymmetry is one of the key ingredients to perform measurements of $CP$ violation in b-hadron decays at the LHC, since $CP$ asymmetries must be disentangled from other sources. The measurements of the production asymmetries are performed in bins of $p_\mathrm{T}$ and $\eta$ of the $B$-meson. The values of the production asymmetries, integrated in the ranges $4 < p_\mathrm{T} < 30$ GeV/c and $2.5<\eta<4.5$, are determined to be: \begin{equation} A_\mathrm{P}(\B^0)= (-1.00\pm0.48\pm0.29)\%,\nonumber \end{equation} \begin{equation} A_\mathrm{P}(\B^0_s)= (\phantom{-}1.09\pm2.61\pm0.61)\%,\nonumber \end{equation} where the first uncertainty is statistical and the second is systematic. The measurement of $A_\mathrm{P}(B^0)$ is performed using the full statistics collected by LHCb so far, corresponding to an integrated luminosity of 3 fb$^{-1}$, while the measurement of $A_\mathrm{P}(B^0_s)$ is realized with the first 1 fb$^{-1}$, leaving room for improvement. No clear evidence of dependences on the values of $p_\mathrm{T}$ and $\eta$ is observed. The results presented in this thesis are the most precise measurements available up to date.

Structural and functional characterization of Group B Streptococcus pilus 2b

Group B Streptococcus (GBS) is a Gram-positive human pathogen representing one of the most common causes of life-threatening bacterial infections such as sepsis and meningitis in neonates. Covalently polymerized pilus-like structures have been discovered in GBS as important virulence factors as well as vaccine candidates. Pili are protein polymers forming long and thin filamentous structures protruding from bacterial cells, mediating adhesion and colonization to host cells. Gram-positive bacteria, including GBS, build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates that are the backbone protein forming the pilus shaft and two ancillary proteins. Also the cell-wall anchoring of the pilus polymers made of covalently linked pilin subunits is mediated by a sortase enzyme. GBS expresses three structurally distinct pilus types (type 1, 2a and 2b). Although the mechanisms of assembly and cell wall anchoring of GBS types 1 and 2a pili have been investigated, those of pilus 2b are not understood until now. Pilus 2b is frequently found in ST-17 strains that are mostly associated with meningitis and high mortality rate especially in infants. In this work the assembly mechanism of GBS pilus type 2b has been elucidated by dissecting through genetic, biochemical and structural studies the role of the two pilus-associated sortases. The most significant findings show that pilus 2b assembly appears “non-canonical”, differing significantly from current pilus assembly models in Gram-positive pathogens. Only sortase-C1 is involved in pilin polymerization, while the sortase-C2 does not act as a pilin polymerase, but it is involved in cell-wall pilus anchoring. Our findings provide new insights into pili biogenesis in Gram-positive bacteria. Moreover, the role of this pilus type during host infection has been investigated. By using a mouse model of meningitis we demonstrated that type 2b pilus contributes to pathogenesis of meningitis in vivo.

Studies towards the total synthesis of penicillipyrone b via a biomimetic approach

Pennicillipyrone A and B are two novel meroterpenoids isolated from the marine-derived fungus Penicilliump sp. Although a preliminary toxicity studies demonstrated the bioactivity of penicillipyrone A to be far superior to that of its congener penicillipyrone B, we were intrigued by its structure. Moreover, it appeared as though one could design an efficient total synthesis based on chemistry that was familiar to our laboratory. The purpose of this project was the study of a new synthesis of Pennicillipyrone B by way of a doubley-biomimetic approach. The intended approach proceeds through a polyene cascade reaction terminated by a nucleophilic pyrone - a reaction not yet known in the literature for the construction of this type of scaffold. During the course of this study we have learned about the unanticipated reactivity of C2 substituted keto-dioxinones with regard to self-condensation. In addition, four new compounds were synthesized and two synthetic routes to the target molecule are presented.

Reflexiones sobre el proceso creador en Antonio Di Benedetto

Este trabajo se propone iluminar, a partir de los datos aportados en diversas entrevistas, algunos aspectos del proceso creador de Antonio Di Benedetto para contribuir a un mayor conocimiento de su poética, especialmente de las reflexiones sobre la creación. Se parte del núcleo autobiográfico para ahondar en los inicios de su actividad de escritor: los años de aprendizaje, la imitación de su madre, una innata narradora, y las influencias de las lecturas de los grandes maestros narradores. A continuación se profundiza en la importancia del silencio en la obra de Di Benedetto, tanto en su función temática como estilística, ya que éste es el núcleo de un decir riguroso, esencial, donde lo no dicho adquiere valor y peso en sí mismo. Finalmente se estudia la estrecha vinculación de sus obras con el particular momento vital, así como su insistente b��squeda de perfección, que lo llevan a explorar las posibilidades expresivas y comunicativas de las diversas modalidades de la ficción, como la narrativa experimental y la fantástica.

Elementos de la vanguardia surrealista en la obra de Antonio Di Benedetto

La complejidad de la literatura del escritor mendocino Antonio Di Benedetto, autor de novelas fundamentales en la literatura latinoamericana como Zama y El silenciero, es abordada, mediante el estudio comparativo, a través de los documentos producidos por el surrealismo. Del vínculo de los relatos del narrador cuyano con la vanguardia se desprende un análisis crítico de la realidad en la obra literaria, como así también de los límites de los postulados del movimiento de 1924 tomados como insumos en la obra de Di Benedetto. Por otro lado, se han estudiado las propuestas del poeta vinculado al surrealismo Antonin Artaud, para relacionarlas con los quiebres discursivos que se repiten a lo largo de la narrativa dibenedettiana.

Kafka en la obra de Antonio di Benedetto

La influencia de Kafka en los escritores argentinos del siglo XX es bien conocida y ha sido extenspmente estudiada especialmente en los autores centrales del canon literario, como son Borges y Sábato. Sin embargo. otros autores en los que se manifiesta dicha influencia son sistemáticamente excluidos de los estudios. Entre ellos se encuentra Antonio Di Benedetto. En el presente trabajo se analiza la presencia de una serie de motivos, caracterizados por la crítica como típicamente kajkianos en la novela más reconocida del mendocino: Zama. De esta manera se apunta a completar el panorama de la recepción de Kafka en la Argentina haciendo hincapié en la experiencia regional, que por su contexto especifico de producción es mucho más afin a la de Kafka que la de los escritores canónicos. Por otra parte, la detección y análisis de esos motivos constituyen una poderosa herramienta para el estudio de la obra dibenedettiana. pues no se trata meramente de la repetición de tópicos consagrados. sino de la manifestación de una experiencia muy personal del mundo.

Annabella (El pentágono) de Antonio Di Benedetto : aproximaciones a una poética de la ficción

A partir de la idea de que las obras literarias pueden instaurar, dentro de su propia estructura, claros y profundos planteos sobre conceptos como realidad, ficción, y verdad ficcional. se indaga la poética de la ficción que de modo implícito se inscribe en la obra de Antonio Di Benedetto, más precisamente en El pentágono; novela en forma de cuentos, texto de corte experimental publicado por vez primera en 1955, en el que se presta especial atención a las relaciones entre realidad y ficción, a la posibilidad de la ficción de instituir mundos diversos y a los límites y juegos especulares entre ambos dominios. Por otra parte, se observa, en la peculiar construcción de esta novela en forma de cuentos, una estructura que en sí misma es signo del concepto de ficción que domina la obra.

Cuerpos invadidos : cuerpo y corporalidad en algunos relatos de Antonio Di Benedetto

Mundo Animal (1953) es una colección de cuentos que inicia la carrera literaria del narrador mendocino, Antonio Di Benedetto. Libro de difícil ubicación genérica, tiene una marcada intencionalidad ética que el mismo autor señala de modo explícito. Es además, evidente la filiación kafkianas de muchas de estas historias. Esta relación se observa, especialmente, en el clima fuertemente onírico y opresivo tanto para los personajes como para los lectores, en el escaso interés por la realidad circundante y en la presencia de un espacio de tipo esquemático, fuertemente simb��lico. Este trabajo se propone, precisamente, detenerse en el análisis de los grietas de este espacio que, si bien no refleja la realidad circundante o referencial, ahonda en torno a otras realidades de profunda relevancia para el hombre contemporáneo. Entre estos espacios esquemáticos, pero de gran valor metafórico, llama la atención la fuerte presencia del cuerpo que, en varios relatos, es presentado como un espacio dentro del cual se desarrolla la acción, o bien que involucra fuertemente dicha acción. En primer lugar estudiaremos la noción de cuerpo y corporalidad presente en el horizonte cultural de Di Benedetto para centrarnos luego en el análisis específico de los cuentos que textualizan el espacio corporal.