Tannerella forsythia and Pseudomonas aeruginosa in subgingival bacterial samples from parous women

BACKGROUND: Information on the subgingival microbiota in parous women is limited. The present study assessed 74 bacterial species at periodontal sites. METHODS: Subgingival bacterial plaque was collected from women > or =6 months after delivery. Bacteria were assessed by the checkerboard DNA-DNA hybridization method. Gingivitis was defined as > or =20% of sites with bleeding on probing (BOP), and periodontitis was defined as radiographic evidence of bone loss and probing depths > or =5.0 mm. RESULTS: A total of 197 women (mean age: 29.4 +/- 6.8 years; range: 18 to 46 years) were included in the study. Gingivitis was identified in 82 of 138 subjects without evidence of periodontitis (59.4%). Periodontitis was found in 59 women (32%). Higher bacterial levels in subjects with gingivitis compared to those without evidence of gingivitis were observed for Actinomyces neuii, Bifidobacterium bifidum, Corynebacterium pseudogenitalis, Porphyromonas endodontalis, Prevotella bivia, and Pseudomonas aeruginosa (P <0.001 for each). Higher bacterial levels in subjects with periodontitis compared to those without periodontitis (BOP not accounted for) were found for 32 of 79 species (P <0.001) including Lactobacillus iners, Haemophilus influenzae, Porphyromonas gingivalis, Tannerella forsythia (previously T. forsythensis), Prevotella bivia, P. aeruginosa, and Staphylococcus aureus. Binary univariate logistic regression analysis identified that P. aeruginosa (P <0.001) and T. forsythia (P <0.05) were independently predictive of periodontal status. The odds ratio of having P. aeruginosa at levels > or =1 x 10(5) in the sample and periodontitis was 3.1 (95% confidence interval: 1.6 to 5.9; P <0.001). CONCLUSION: In addition to P. gingivalis and T. forsythia, a diverse microbiota, including P. aeruginosa, P. endodontalis, P. bivia, and S. aureus, can be found in subgingival plaque samples from women of child-bearing age with periodontitis.

The impact of the stone age diet on gingival conditions in the absence of oral hygiene

BACKGROUND: The objective of this study was to assess the oral microbiota and clinical data in subjects without access to traditional oral hygiene methods and who ate a diet available in the Stone Age. METHODS: Ten subjects living in an environment replicating the Stone Age for 4 weeks were enrolled in this study. Bleeding on probing (BOP), gingival and plaque indices, and probing depth (PD) were assessed at baseline and at 4 weeks. Microbiologic samples were collected at the mesio-buccal subgingival aspects of all teeth and from the dorsum of the tongue and were processed by checkerboard DNA-DNA hybridization methods. RESULTS: No subject had periodontitis. Mean BOP decreased from 34.8% to 12.6% (P <0.001). Mean gingival index scores changed from 0.38 to 0.43 (not statistically significant) and mean plaque scores increased from 0.68 to 1.47 (P <0.001). PD at sites of subgingival sampling decreased (mean difference: 0.2 mm; P <0.001). At week 4, the total bacterial count was higher (P <0.001) for 24 of 74 species, including Bacteroides ureolyticus, Eikenella corrodens, Lactobacillus acidophilus, Capnocytophaga ochracea, Escherichia coli, Fusobacterium nucleatum naviforme, Haemophilus influenzae, Helicobacter pylori, Porphyromonas endodontalis, Staphylococcus aureus (two strains), Streptococcus agalactiae, Streptococcus anginosis, and Streptococcus mitis. Bacterial counts from tongue samples were higher at baseline (P <0.001) for 20 species, including Tannerella forsythia (previously T. forsythensis), Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans; serotype a), and Streptococcus spp. CONCLUSIONS: The experimental gingivitis protocol is not applicable if the diet (e.g., Stone Age) does not include refined sugars. Although plaque levels increased, BOP and PD decreased. Subgingival bacterial counts increased for several species not linked to periodontitis, whereas tongue bacterial samples decreased during the study period.

Cloning and characterization of an Actinobacillus pleuropneumoniae outer membrane protein belonging to the family of PAL lipoproteins

A 14-kDa outer membrane protein (OMP) was purified from Actinobacillus pleuro-pneumoniae serotype 2. The protein strongly reacts with sera from pigs experimentally or naturally infected with any of the 12 serotypes of A. pleuropneumoniae. The gene encoding this protein was isolated from a gene library of A. pleuropneumoniae serotype 2 reference strain by immunoscreening. Expression of the cloned gene in Escherichia coli revealed that the protein is also located in the outer membrane fraction of the recombinant host. DNA sequence analysis of the gene reveals high similarity of the protein's amino acid sequence to that of the E. coli peptidoglycan-associated lipoprotein PAL, to the Haemophilus influenzae OMP P6 and to related proteins of several other Gram-negative bacteria. We have therefore named the 14-kDa protein PalA, and its corresponding gene, palA. The 20 amino-terminal amino acid residues of PalA constitute a signal sequence characteristic of membrane lipoproteins of prokaryotes with a recognition site for the signal sequence peptidase II and a sorting signal for the final localization of the mature protein in the outer membrane. The DNA sequence upstream of palA contains an open reading frame which is highly similar to the E. coli tolB gene, indicating a gene cluster in A. pleuropneumoniae which is very similar to the E. coli tol locus. The palA gene is conserved and expressed in all A. pleuropneumoniae serotypes and in A. lignieresii. A very similar palA gene is present in A. suis and A. equuli.

Cluster of Bacteria Associated with Peri-Implantitis.

BACKGROUND Information on the microbiota in peri-implantitis is limited. We hypothesized that neither gender nor a history of periodontitis/smoking or the microbiota at implants differ by implant status. MATERIALS AND METHODS Baseline microbiological samples collected at one implant in each of 166 participants with peri-implantitis and from 47 individuals with a healthy implant were collected and analyzed by DNA-DNA checkerboard hybridization (78 species). Clinical and radiographic data defined implant status. RESULTS Nineteen bacterial species were found at higher counts from implants with peri-implantitis including Aggregatibacter actinomycetemcomitans, Campylobacter gracilis, Campylobacter rectus, Campylobacter showae, Helicobacter pylori, Haemophilus influenzae, Porphyromonas gingivalis, Staphylococcus aureus, Staphylococcus anaerobius, Streptococcus intermedius, Streptococcus mitis, Tannerella forsythia, Treponema denticola, and Treponema socranskii (p < .001). Receiver operating characteristic curve analysis identified T. forsythia, P. gingivalis, T. socranskii, Staph. aureus, Staph. anaerobius, Strep. intermedius, and Strep. mitis in peri-implantitis comprising 30% of the total microbiota. When adjusted for gender (not significant [NS]), smoking status (NS), older age (p = .003), periodontitis history (p < .01), and T. forsythia (likelihood ratio 3.6, 95% confidence interval 1.4, 9.1, p = .007) were associated with peri-implantitis. CONCLUSION A cluster of bacteria including T. forsythia and Staph. aureus are associated with peri-implantitis.

The Family Pasteurellaceae

This chapter describes the systematics and evolution of Pasteurellaceae with emphasis on new information generated since the 3rd edition of The Prokaryotes which only included chapters dealing with Haemophilus, Actinobacillus, and Pasteurella. A major source of new information for the current chapter has been provided by whole genome sequences now available for many taxa of the family. Some 100 species and species-like taxa have been documented and 18 genera of Pasteurellaceae reported so far. Members of the family include specialized commensals, potential pathogens, or pathogens of vertebrates and mainly survive poorly in other habitats including the external environment. The pathogenic members are of major importance to animal production and human health. Members of Pasteurellaceae have relatively small genomes, probably as a result of adaption to a special habitat. The most important species in veterinary microbiology include Pasteurella multocida, Actinobacillus pleuropneumoniae, [Haemophilus] parasuis, Mannheimia haemolytica, Bibersteinia trehalosi, and Avibacterium paragallinarum, while Haemophilus influenzae and Aggregatibacter actinomycetemcomitans represent the most important species as to human disease. Traditional isolation techniques are still used in both human and veterinary clinical diagnostic laboratories although genetically based diagnostic methods have replaced traditional biochemical/physiological methods for characterization and identification. For all species, MALDI-TOF can now be used as a diagnostic tool. As control and if MALDI-TOF equipment is not at hand, PCR-based specific detection is possible for Pasteurella multocida, Actinobacillus pleuropneumoniae, [Haemophilus] parasuis, Mannheimia haemolytica, Avibacterium paragallinarum, Gallibacterium anatis, Haemophilus influenzae, and Aggregatibacter actinomycetemcomitans. A lot of work has been directed towards identification of virulence factors and understanding host microbe interactions involved in disease.

Reclassification of Actinobacillus muris as Muribacter muris gen. nov. comb. nov.

To reinvestigate the taxonomy of [Actinobacillus] muris, 474 strains mainly from mice and rats were characterized by phenotype and 130 strains selected for genotypic characterization by 16S rRNA and partial rpoB gene sequencing. The type strain was further investigated by whole genome sequencing. Phylogenetic analysis of the DNA sequences showed one monophyletic group with intra group similarities of 96.7 % and 97.2 % for 16S rRNA and rpoB genes, respectively. The lowest 16S rRNA similarity to the closest related valid named taxon outside the group was 95.9 % to the type strain of [Pasteurella] pneumotropica. The closest related taxon based on rpoB sequence comparison was 'Haemophilus influenzae-murium' with 88.4 %. A new genus, Muribacter is proposed based on a distinct phylogenetic position based on 16S rRNA and rpoB gene sequence comparisons with major divergence to the existing genera of Pasteurellaceae. The new genus includes the characteristics of [Actinobacillus] muris with the emendation that acid formation from (-)-D-mannitol is variable as well the hydrolysis of esculin while the α-glucosidase test is positive. There is no requirement for exogenously supplied nicotinamide adenine dinucleotide (V factor) for the majority of strains investigated, however, one strain was found positive. The major fatty acids of the type strain of Muribacter muris were C 14:0, C 14:0 3OH/C 16:1 ISOI, C 16:1 ω7c and C 16:0 which is in line with most genera of Pasteurellaceae. The type strain of Muribacter muris is CCUG 16938T ( = NCTC 12432T = ATCC 49577T).

Survey of VFC provider knowledge of catch-up regimens and contraindications to vaccination in Houston, Texas

In 2004, Houston had one of the lowest childhood immunization levels among major metropolitan cities in the United States at 65% for the 4:3:1:3:3 vaccination series. Delays in the receipt of scheduled vaccinations may be related to missed opportunities due to health care provider lack of knowledge about catch-up regimens and contraindications for pediatric vaccination. The objectives of this study are to identify, measure, and report on VFC provider-practice characteristics, knowledge of catch-up regimens and contraindications, and use of Reminder recall (R/R) and moved or gone elsewhere (MOGE) practices among providers with high (>80%) and low (<70%) immunization coverage among 19-35 month old children. The sampling frame consists of 187 Vaccines for Children (VFC) providers with 2004 clinic assessment software application (CASA) scores. Data were collected by personal interview with each participating practice provider. Only ten VFC providers were successful at maximizing vaccinations for every vignette and no provider administered the maximum possible number of vaccinations at visit 2 for all six vignettes. Both coverage groups administered polio conjugate vaccine (PCV), haemophilus influenza type b (Hib), and diphtheria, tetanus and acellular pertussis (DTaP) most frequently and omitted most frequently varicella zoster vaccine (VZV) and measles, mumps, and rubella (MMR) vaccine. ^

Function of C1A barley peptidases and their inhibitors (cystatins) in barley seed germination : Funcion de las peptidasas C1A de cebada y sus inhibidores (cistatinas) en la germinacion de la semilla

Plant proteolysis is a metabolic process where specific enzymes called peptidases degrade proteins. In plants, this complex process involves broad metabolic networks and different sub-cellular compartments. Several types of peptidases take part in the proteolytic process, mainly cysteine-, serine-, aspartyl- and metallo- peptidases. Among the cysteine-peptidases, the papain-like or C1A peptidases (family C1, clan CA) are extensively present in land plants and are classified into catepsins L-, B-, H- and Flike. The catalytic mechanism of these C1A peptidases is highly conserved and involves the three amino acids Cys, His and Asn in the catalytic triad, and a Gln residue which seems essential for maintaining an active enzyme conformation. These proteins are synthesized as inactive precursors, which comprise an N-terminal signal peptide, a propeptide, and the mature protein. In barley, we have identified 33 cysteine-peptidases from the papain-like family, classifying them into 8 different groups. Five of them corresponded to cathepsins L-like (5 subgroups), 1 cathepsin B-like group, 1 cathepsin F-like group and 1 cathepsin H-like group. Besides, C1A peptidases are the specific targets of the plant proteinaceous inhibitors known as phytocystatins (PhyCys). The cystatin inhibitory mechanism is produced by a tight and reversible interaction with their target enzymes. In barley, the cystatin gene family is comprised by 13 members. In this work we have tried to elucidate the role of the C1A cysteine-peptidases and their specific inhibitors (cystatins) in the germination process of the barley grain. Therefore, we selected a representative member of each group/subgroup of C1A peptidases (1 cathepsin B-like, 1 cathepsin F-like, 1 cathepsin H-like and 5 cathepsins L-like). The molecular characterization of the cysteine-peptidases was done and the peptidase-inhibitor interaction was analyzed in vitro and in vivo. A study in the structural basis for specificity of pro-peptide/enzyme interaction in barley C1A cysteine-peptidases has been also carried out by inhibitory assays and the modeling of the three-dimensional structures. The barley grain maturation produces the accumulation of storage proteins (prolamins) in the endosperm which are mobilized during germination to supply the required nutrients until the photosynthesis is fully established. In this work, we have demonstrated the participation of the cysteine-peptidases and their inhibitors in the degradation of the different storage protein fractions (hordeins, albumins and globulins) present in the barley grain. Besides, transgenic barley plants overexpressing or silencing cysteine-peptidases or cystatins were obtained by Agrobacterium-mediated transformation of barley immature embryos to analyze their physiological function in vivo. Preliminary assays were carried out with the T1 grains of several transgenic lines. Comparing the knock-out and the overexpressing lines with the WT, alterations in the germination process were detected and were correlated with their grain hordein content. These data will be validated with the homozygous grains that are being produced through the double haploid technique by microspore culture. Resumen La proteólisis es un proceso metabólico por el cual se lleva a cabo la degradación de las proteínas de un organismo a través de enzimas específicas llamadas proteasas. En plantas, este complejo proceso comprende un entramado de rutas metabólicas que implican, además, diferentes compartimentos subcelulares. En la proteólisis participan numerosas proteasas, principalmente cisteín-, serín-, aspartil-, y metalo-proteasas. Dentro de las cisteín-proteasas, las proteasas tipo papaína o C1A (familia C1, clan CA) están extensamente representadas en plantas terrestres, y se clasifican en catepsinas tipo L, B, H y F. El mecanismo catalítico de estas proteasas está altamente conservado y la triada catalítica formada por los aminoácidos Cys, His y Asn, y a un aminoácido Gln, que parece esencial para el mantenimiento de la conformación activa de la proteína. Las proteasas C1A se sintetizan como precursores inactivos y comprenden un péptido señal en el extremo N-terminal, un pro-péptido y la proteína madura. En cebada hemos identificado 33 cisteín-proteasas de tipo papaína y las hemos clasificado filogenéticamente en 8 grupos diferentes. Cinco de ellos pertenecen a las catepsinas tipo L (5 subgrupos), un grupo a las catepsinas tipo-B, otro a las catepsinas tipo-F y un último a las catepsinas tipo-H. Las proteasas C1A son además las dianas específicas de los inhibidores protéicos de plantas denominados fitocistatinas. El mecanismo de inhibición de las cistatinas está basado en una fuerte interacción reversible. En cebada, se conoce la familia génica completa de las cistatinas, que está formada por 13 miembros. En el presente trabajo se ha investigado el papel de las cisteín-proteasas de cebada y sus inhibidores específicos en el proceso de la germinación de la semilla. Para ello, se seleccionó una proteasa representante de cada grupo/subgrupo (1 catepsina tipo- B, 1 tipo-F, 1 tipo-H, y 5 tipo-L, una por cada subgrupo). Se ha llevado a cabo su caracterización molecular y se ha analizado la interacción enzima-inhibidor tanto in vivo como in vitro. También se han realizado estudios sobre las bases estructurales que demuestran la especificidad en la interacción enzima/propéptido en las proteasas C1A de cebada, mediante ensayos de inhibición y la predicción de modelos estructurales de la interacción. Finalmente, y dado que durante la maduración de la semilla se almacenan proteínas de reserva (prolaminas) en el endospermo que son movilizadas durante la germinación para suministrar los nutrientes necesarios hasta que la nueva planta pueda realizar la fotosíntesis, en este trabajo se ha demostrado la participación de las cisteínproteasas y sus inhibidores en la degradación de las diferentes tipos de proteínas de reserva (hordeinas, albúmins y globulinas) presentes en el grano de cebada. Además, se han obtenido plantas transgénicas de cebada que sobre-expresan o silencian cistatinas y cisteín-proteasas con el fin de analizar la función fisiológica in vivo. Se han realizado análisis preliminares en las semillas T1 de varias líneas tránsgenicas de cebada y al comparar las líneas knock-out y las líneas de sobre-expresión con las silvestres, se han detectado alteraciones en la germinación que están además correlacionadas con el contenido de hordeinas de las semillas. Estos datos serán validados en las semillas homocigotas que se están generando mediante la técnica de dobles haploides a partir del cultivo de microesporas.

Estudio de la rotura en barras de acero : aspectos experimentales y numéricos

El acero es, junto con el hormigón, el material más ampliamente empleado en la construcción de obra civil y de edificación. Además de su elevada resistencia, su carácter dúctil resulta un aspecto de particular interés desde el punto de vista de la seguridad estructural, ya que permite redistribuir esfuerzos a elementos adyacentes y, por tanto, almacenar una mayor energía antes del colapso final de la estructura. No obstante, a pesar de su extendida utilización, todavía existen aspectos relacionados con su comportamiento en rotura que necesitan una mayor clarificación y que permitirían un mejor aprovechamiento de sus propiedades. Cuando un elemento de acero es ensayado a tracción y alcanza la carga máxima, sufre la aparición de un cuello de estricción que plantea dificultades para conocer el comportamiento del material desde dicho instante hasta la rotura. La norma ISO 6892-1, que define el método a emplear en un ensayo de tracción con materiales metálicos, establece procedimientos para determinar los parámetros relacionados con este tramo último de la curva F − E. No obstante, la definición de dichos parámetros resulta controvertida, ya que éstos presentan una baja reproducibilidad y una baja repetibilidad que resultan difíciles de explicar. En esta Tesis se busca profundizar en el conocimiento del último tramo de la curva F − E de los aceros de construcción. Para ello se ha realizado una amplia campaña experimental sobre dos aceros representativos en el campo de la construcción civil: el alambrón de partida empleado en la fabricación de alambres de pretensado y un acero empleado como refuerzo en hormigón armado. Los dos materiales analizados presentan formas de rotura diferentes: mientras el primero de ellos presenta una superficie de rotura plana con una región oscura claramente apreciable en su interior, el segundo rompe según la clásica superficie en forma de copa y cono. La rotura en forma de copa y cono ha sido ampliamente estudiada en el pasado y existen modelos de rotura que han logrado reproducirla con éxito, en especial el modelo de Gurson- Tvergaard-Needleman (GTN). En cuanto a la rotura exhibida por el primer material, en principio nada impide abordar su reproducción numérica con un modelo GTN, sin embargo, las diferencias observadas entre ambos materiales en los ensayos experimentales permiten pensar en otro criterio de rotura. En la presente Tesis se realiza una amplia campaña experimental con probetas cilíndricas fabricadas con dos aceros representativos de los empleados en construcción con comportamientos en rotura diferentes. Por un lado se analiza el alambrón de partida empleado en la fabricación de alambres de pretensado, cuyo frente de rotura es plano y perpendicular a la dirección de aplicación de la carga con una región oscura en su interior. Por otro lado, se estudian barras de acero empleadas como armadura pasiva tipo B 500 SD, cuyo frente de rotura presenta la clásica superficie en forma de copa y cono. Estos trabajos experimentales han permitido distinguir dos comportamientos en rotura claramente diferenciados entre ambos materiales y, en el caso del primer material, se ha identificado un comportamiento asemejable al exhibido por materiales frágiles. En este trabajo se plantea la hipótesis de que el primer material, cuya rotura provoca un frente de rotura plano y perpendicular a la dirección de aplicación de la carga, rompe de manera cuasifrágil como consecuencia de un proceso de decohesión, de manera que la región oscura que se observa en el centro del frente de rotura se asemeja a una entalla circular perpendicular a la dirección de aplicación de la carga. Para la reproducción numérica de la rotura exhibida por el primer material, se plantea un criterio de rotura basado en un modelo cohesivo que, como aspecto novedoso, se hace depender de la triaxialidad de tensiones, parámetro determinante en el fallo de este tipo de materiales. Este tipo de modelos presenta varias ventajas respecto a los modelos GTN habitualmente empleados. Mientras los modelos GTN precisan de numerosos parámetros para su calibración, los modelos cohesivos precisan fundamentalmente de dos parámetros para definir su curva de ablandamiento: la tensión de decohesión ft y la energía de fractura GF . Además, los parámetros de los modelos GTN no son medibles de manera experimental, mientras que GF sí lo es. En cuanto a ft, aunque no existe un método para su determinación experimental, sí resulta un parámetro más fácilmente interpretable que los empleados por los modelos GTN, que utilizan valores como el porcentaje de huecos presentes en el material para iniciar el fenómeno de coalescencia o el porcentaje de poros que provoca una pérdida total de la capacidad resistente. Para implementar este criterio de rotura se ha desarrollado un elemento de intercara cohesivo dependiente de la triaxialidad de tensiones. Se han reproducido con éxito los ensayos de tracción llevados a cabo en la campaña experimental empleando dicho elemento de intercara. Además, en estos modelos la rotura se produce fenomenológicamente de la misma manera observada en los ensayos experimentales: produciéndose una decohesión circular en torno al eje de la probeta. En definitiva, los trabajos desarrollados en esta Tesis, tanto experimentales como numéricos, contribuyen a clarificar el comportamiento de los aceros de construcción en el último tramo de la curva F − E y los mecanismos desencadenantes de la rotura final del material, aspecto que puede contribuir a un mejor aprovechamiento de las propiedades de estos aceros en el futuro y a mejorar la seguridad de las estructuras construidas con ellos. Steel is, together with concrete, the most widely used material in civil engineering works. Not only its high strength, but also its ductility is of special interest from the point of view of the structural safety, since it enables stress distribution with adjacent elements and, therefore, more energy can be stored before reaching the structural failure. However, despite of being extensively used, there are still some aspects related to its fracture behaviour that need to be clarified and that will allow for a better use of its properties. When a steel item is tested under tension and reaches the maximum load point, necking process begins, which makes difficult to define the material behaviour from that moment onward. The ISO standard 6892-1, which defines the tensile testing method for metallic materials, describes the procedures to obtain some parameters related to this last section of the F − E curve. Nevertheless, these parameters have proved to be controversial, since they have low reproducibility and repeatibility rates that are difficult to explain. This Thesis tries to deepen the knowledge of the last section of the F − E curve for construction steels. An extensive experimental campaign has been carried out with two representative steels used in civil engineering works: a steel rod used for manufacturing prestressing steel wires, before the cold-drawing process is applied, and steel bars used in reinforced concrete structures. Both materials have different fracture surfaces: while the first of them shows a flat fracture surface, perpendicular to the loading direction with a dark region in the centre of it, the second one shows the classical cup-cone fracture surface. The cup-cone fracture surface has been deeply studied in the past and different numerical models have been able to reproduce it with success, with a special mention to the Gurson-Tvergaard-Needleman model (GTN). Regarding the failure surface shown by the first material, in principle it can be numerically reproduced by a GTN model, but the differences observed between both materials in the experimental campaign suggest thinking of a different failure criterium. In the present Thesis, an extensive experimental campaign has been carried out using cylindrical specimens made of two representative construction steels with different fracture behaviours. On one hand, the initial eutectoid steel rod used for manufacturing prestressing steel wires is analysed, which presents a flat fracture surface, perpendicular to the loading direction, and with a dark region in the centre of it. On the other hand, B 500 SD steel bars, typically used in reinforced concrete structures and with the typical cup-cone fracture surface, are studied. These experimental works have allowed distinguishing two clearly different fracture behaviours between both materials and, in the case of the first one, a fragile-like behaviour has been identified. For the first material, which shows a flat fracture surface perpendicular to the loading direction, the following hypothesis is proposed in this study: a quasi-brittle fracture is developed as a consequence of a decohesion process, with the dark region acting as a circular crack perpendicular to the loading direction. To reproduce numerically the fracture behaviour shown by the first material, a failure criterium based on a cohesive model is proposed in this Thesis. As an innovative contribution, this failure criterium depends on the stress triaxiality state of the material, which is a key parameter when studying fracture in this kind of materials. This type of models have some advantages when compared to the widely used GTN models. While GTN models need a high number of parameters to be defined, cohesive models need basically two parameters to define the softening curve: the decohesion stress ft and the fracture energy GF . In addition to this, GTN models parameters cannot be measured experimentally, while GF is indeed. Regarding ft, although no experimental procedure is defined for its obtention, it has an easier interpretation than the parameters used by the GTN models like, for instance, the void volume needed for the coalescence process to start or the void volume that leads to a total loss of the bearing capacity. In order to implement this failure criterium, a triaxiality-dependent cohesive interface element has been developed. The experimental results obtained in the experimental campaign have been successfully reproduced by using this interface element. Furthermore, in these models the failure mechanism is developed in the same way as observed experimentally: with a circular decohesive process taking place around the longitudinal axis of the specimen. In summary, the works developed in this Thesis, both experimental and numerical, contribute to clarify the behaviour of construction steels in the last section of the F − E curve and the mechanisms responsible for the eventual material failure, an aspect that can lead to a better use of the properties of these steels in the future and a safety improvement in the structures built with them.

Estudio del comportamiento en rotura de un material metálico dúctil : aspectos experimentales y numéricos

La motivación principal de este trabajo fin de máster es el estudio del comportamiento en rotura de un material metálico muy dúctil como es una aleación de aluminio. El conocimiento del comportamiento de los materiales en su régimen plástico es muy valioso, puesto que el concepto de ductilidad de un material está relacionado directamente con la seguridad de una estructura. Un material dúctil es aquel que resiste estados tensionales elevados y alcanza altos niveles de deformación, siendo capaz de absorber gran cantidad de energía antes de su rotura y permitiendo una redistribución de esfuerzos entre elementos estructurales. Por tanto, la utilización de materiales dúctiles en el mundo de la construcción supone en general un incremento de la seguridad estructural por su “capacidad de aviso”, es decir, la deformación que estos materiales experimentan antes de su rotura. Al contrario que los materiales frágiles, que carecen de esta capacidad de aviso antes de su rotura, produciéndose ésta de forma repentina y sin apenas deformación previa. En relación a esto, el ensayo de tracción simple se considera una de las técnicas más sencillas y utilizadas en la caracterización de materiales metálicos, puesto que a partir de la curva fuerza-desplazamiento que este ensayo proporciona, permite obtener de forma precisa la curva tensión-deformación desde el instante de carga máxima. No obstante, existen dificultades para la definición del comportamiento del material desde el instante de carga máxima hasta rotura, lo que provoca que habitualmente no se considere este último tramo de la curva tensión-deformación cuando, tal y como sabemos, contiene una información muy importante y valiosa. Y es que, este último tramo de la curva tensión-deformación es primordial a la hora de determinar la energía máxima que un elemento es capaz de absorber antes de su rotura, aspecto elemental, por ejemplo para conocer si una rotura ha sido accidental o intencionada. Por tanto, el tramo final de la curva tensión-deformación proporciona información muy interesante sobre el comportamiento del material frente a situaciones límite de carga. El objetivo por tanto va a ser continuar con el trabajo realizado por el doctor Ingeniero de Caminos, Canales y Puertos, Fernando Suárez Guerra, el cual estudió en su Tesis Doctoral el comportamiento en rotura de dos materiales metálicos como son, un Material 1: acero perlítico empleado en la fabricación de alambres de pretensado, y un Material 2: acero tipo B 500 SD empleado como armadura pasiva en hormigón armado. Estos materiales presentan un comportamiento a rotura claramente diferenciado, siendo más dúctil el Material 2 que el Material 1. Tomando como partida esta Tesis Doctoral, este Trabajo Fin de Máster pretende continuar con el estudio del comportamiento en rotura de un material metálico mucho más dúctil que los experimentados anteriormente, como es el aluminio. Analizando el último tramo de la curva tensión-deformación, que corresponde al tramo entre el instante de carga máxima y el de rotura del material. Atendiendo a los mecanismos de rotura de un material metálico, es necesario distinguir dos comportamientos distintos. Uno que corresponde a una rotura en forma de copa y cono, y otro que corresponde a una superficie de rotura plana perpendicular a la dirección de aplicación de la carga.

Pulsatile blood flow interaction with arterial walls of aorta : autoregulation and impedance pressure boundary condition and its biomedical applications

Para las decisiones urgentes sobre intervenciones quirúrgicas en el sistema cardiovascular se necesitan simulaciones computacionales con resultados fiables y que consuman un tiempo de cálculo razonable. Durante años los investigadores han trabajado en diversos métodos numéricos de cálculo que resulten atractivos para los cirujanos. Estos métodos, precisos pero costosos desde el punto de vista del coste computacional, crean un desajuste entre la oferta de los ingenieros que realizan las simulaciones y los médicos que operan en el quirófano. Por otra parte, los métodos de cálculo más simplificados reducen el tiempo de cálculo pero pueden proporcionar resultados no realistas. El objetivo de esta tesis es combinar los conceptos de autorregulación e impedancia del sistema circulatorio, la interacción flujo sanguíneo-pared arterial y modelos geométricos idealizados tridimensionales de las arterias pero sin pérdida de realismo, con objeto de proponer una metodología de simulación que proporcione resultados correctos y completos, con tiempos de cálculo moderados. En las simulaciones numéricas, las condiciones de contorno basadas en historias de presión presentan inconvenientes por ser difícil conocerlas con detalle, y porque los resultados son muy sensibles ante pequeñas variaciones de dichas historias. La metodología propuesta se basa en los conceptos de autorregulación, para imponer la demanda de flujo aguas abajo del modelo en el ciclo cardiaco, y la impedancia, para representar el efecto que ejerce el flujo en el resto del sistema circulatorio sobre las arterias modeladas. De este modo las historias de presión en el contorno son resultados del cálculo, que se obtienen de manera iterativa. El método propuesto se aplica en una geometría idealizada del arco aórtico sin patologías y en otra geometría correspondiente a una disección Stanford de tipo A, considerando la interacción del flujo pulsátil con las paredes arteriales. El efecto de los tejidos circundantes también se incorpora en los modelos. También se hacen aplicaciones considerando la interacción en una geometría especifica de un paciente anciano que proviene de una tomografía computarizada. Finalmente se analiza una disección Stanford tipo B con tres modelos que incluyen la fenestración del saco. Clinicians demand fast and reliable numerical results of cardiovascular biomechanic simulations for their urgent pre-surgery decissions. Researchers during many years have work on different numerical methods in order to attract the clinicians' confidence to their colorful contours. Though precise but expensive and time-consuming methodologies create a gap between numerical biomechanics and hospital personnel. On the other hand, simulation simplifications with the aim of reduction in computational time may cause in production of unrealistic outcomes. The main objective of the current investigation is to combine ideas such as autoregulation, impedance, fluid-solid interaction and idealized geometries in order to propose a computationally cheap methodology without excessive or unrealistic simplifications. The pressure boundary conditions are critical and polemic in numerical simulations of cardiovascular system, in which a specific arterial site is of interest and the rest of the netwrok is neglected but represented by a boundary condition. The proposed methodology is a pressure boundary condition which takes advantage of numerical simplicity of application of an imposed pressure boundary condition on outlets, while it includes more sophisticated concepts such as autoregulation and impedance to gain more realistic results. Incorporation of autoregulation and impedance converts the pressure boundary conditions to an active and dynamic boundary conditions, receiving feedback from the results during the numerical calculations and comparing them with the physiological requirements. On the other hand, the impedance boundary condition defines the shapes of the pressure history curves applied at outlets. The applications of the proposed method are seen on idealized geometry of the healthy arotic arch as well as idealized Stanford type A dissection, considering the interaction of the arterial walls with the pulsatile blood flow. The effect of surrounding tissues is incorporated and studied in the models. The simulations continue with FSI analysis of a patient-specific CT scanned geometry of an old individual. Finally, inspiring of the statistic results of mortality rates in Stanford type B dissection, three models of fenestrated dissection sac is studied and discussed. Applying the developed boundary condition, an alternative hypothesis is proposed by the author with respect to the decrease in mortality rates in patients with fenestrations.

The identification of CD4+ T cell epitopes with dedicated synthetic peptide libraries

For a large number of T cell-mediated immunopathologies, the disease-related antigens are not yet identified. Identification of T cell epitopes is of crucial importance for the development of immune-intervention strategies. We show that CD4+ T cell epitopes can be defined by using a new system for synthesis and screening of synthetic peptide libraries. These libraries are designed to bind to the HLA class II restriction molecule of the CD4+ T cell clone of interest. The screening is based on three selection rounds using partial release of 14-mer peptides from synthesis beads and subsequent sequencing of the remaining peptide attached to the bead. With this approach, two peptides were identified that stimulate the β cell-reactive CD4+ T cell clone 1c10, which was isolated from a newly diagnosed insulin-dependent diabetes mellitus patient. After performing amino acid-substitution studies and protein database searches, a Haemophilus influenzae TonB-derived peptide was identified that stimulates clone 1c10. The relevance of this finding for the pathogenesis of insulin-dependent diabetes mellitus is currently under investigation. We conclude that this system is capable of determining epitopes for (autoreactive) CD4+ T cell clones with previously unknown peptide specificity. This offers the possibility to define (auto)antigens by searching protein databases and/or to induce tolerance by using the peptide sequences identified. In addition the peptides might be used as leads to develop T cell receptor antagonists or anergy-inducing compounds.

Identification of a novel selD homolog from Eukaryotes, Bacteria, and Archaea: Is there an autoregulatory mechanism in selenocysteine metabolism?

Escherichia coli selenophosphate synthetase (SPS, the selD gene product) catalyzes the production of monoselenophosphate, the selenium donor compound required for synthesis of selenocysteine (Sec) and seleno-tRNAs. We report the molecular cloning of human and mouse homologs of the selD gene, designated Sps2, which contains an in-frame TGA codon at a site corresponding to the enzyme’s putative active site. These sequences allow the identification of selD gene homologs in the genomes of the bacterium Haemophilus influenzae and the archaeon Methanococcus jannaschii, which had been previously misinterpreted due to their in-frame TGA codon. Sps2 mRNA levels are elevated in organs previously implicated in the synthesis of selenoproteins and in active sites of blood cell development. In addition, we show that Sps2 mRNA is up-regulated upon activation of T lymphocytes and have mapped the Sps2 gene to mouse chromosome 7. Using the mouse gene isolated from the hematopoietic cell line FDCPmixA4, we devised a construct for protein expression that results in the insertion of a FLAG tag sequence at the N terminus of the SPS2 protein. This strategy allowed us to document the readthrough of the in-frame TGA codon and the incorporation of 75Se into SPS2. These results suggest the existence of an autoregulatory mechanism involving the incorporation of Sec into SPS2 that might be relevant to blood cell biology. This mechanism is likely to have been present in ancient life forms and conserved in a variety of living organisms from all domains of life.

Evolutionary relationships among diverse bacteriophages and prophages: All the world’s a phage

We report DNA and predicted protein sequence similarities, implying homology, among genes of double-stranded DNA (dsDNA) bacteriophages and prophages spanning a broad phylogenetic range of host bacteria. The sequence matches reported here establish genetic connections, not always direct, among the lambdoid phages of Escherichia coli, phage φC31 of Streptomyces, phages of Mycobacterium, a previously unrecognized cryptic prophage, φflu, in the Haemophilus influenzae genome, and two small prophage-like elements, φRv1 and φRv2, in the genome of Mycobacterium tuberculosis. The results imply that these phage genes, and very possibly all of the dsDNA tailed phages, share common ancestry. We propose a model for the genetic structure and dynamics of the global phage population in which all dsDNA phage genomes are mosaics with access, by horizontal exchange, to a large common genetic pool but in which access to the gene pool is not uniform for all phage.

Genetic footprinting with mariner-based transposition in Pseudomonas aeruginosa

The complete DNA sequence of Pseudomonas aeruginosa provides an opportunity to apply functional genomics to a major human pathogen. A comparative genomics approach combined with genetic footprinting was used as a strategy to identify genes required for viability in P. aeruginosa. Use of a highly efficient in vivo mariner transposition system in P. aeruginosa facilitated the analysis of candidate genes of this class. We have developed a rapid and efficient allelic exchange system by using the I-SceI homing endonuclease in conjunction with in vitro mariner mutagenesis to generate mutants within targeted regions of the P. aeruginosa chromosome for genetic footprinting analyses. This technique for generating transposon insertion mutants should be widely applicable to other organisms that are not naturally transformable or may lack well developed in vivo transposition systems. We tested this system with three genes in P. aeruginosa that have putative essential homologs in Haemophilus influenzae. We show that one of three H. influenzae essential gene homologs is needed for growth in P. aeruginosa, validating the practicality of this comparative genomics strategy to identify essential genes in P. aeruginosa.