Clostridium difficile is an obligate anaerobic, Gram-positive, endospore-forming bacterium. Although an opportunistic pathogen, it is one of the important causes of healthcare-associated infections. While toxins TcdA and TcdB are the main virulence factors of C. difficile, the factors or processes involved in gut colonization during infection remain unclear. The biofilm-forming ability of bacterial pathogens has been associated with increased antibiotic resistance and chronic recurrent infections. Little is known about biofilm formation by anaerobic gut species. Biofilm formation by C. difficile could play a role in virulence and persistence of C. difficile, as seen for other intestinal pathogens. We demonstrate that C. difficile clinical strains, 630, and the strain isolated in the outbreak, R20291, form structured biofilms in vitro. Biofilm matrix is made of proteins, DNA and polysaccharide. Strain R20291 accumulates substantially more biofilm. Employing isogenic mutants, we show that virulence-associated proteins, Cwp84, flagella and a putative quorum sensing regulator, LuxS, Spo0A, are required for maximal biofilm formation by C. difficile. Moreover we demonstrate that bacteria in C. difficile biofilms are more resistant to high concentrations of vancomycin, a drug commonly used for treatment of CDI, and that inhibitory and sub-inhibitory concentrations of the same antibiotic induce biofilm formation. Surprisingly, clinical C. difficile strains from the same out-break, but from different origin, show differences in biofilm formation. Genome sequence analysis of these strains showed presence of a single nucleoide polymorphism (SNP) in the anti-σ factor RsbW, which regulates the stress-induced alternative sigma factor B (σB). We further demonstrate that RsbW, a negative regulator of alternative sigma factor B, has a role in biofilm formation and sporulation of C. difficile. Our data suggest that biofilm formation by C. difficile is a complex multifactorial process and may be a crucial mechanism for clostridial persistence in the host.

Streptococcus agalactiae, also known as Group B Streptococcus (GBS) is the primary colonizer of the anogenital mucosa of up to 40% of healthy women and an important cause of invasive neonatal infections worldwide. Among the 10 known capsular serotypes, GBS type III accounts for 30-76% of the cases of neonatal meningitis. Biofilms are dense aggregates of surface-adherent microorganisms embedded in an exopolysaccharide matrix. Centers for Disease Control and Prevention estimate that 65% of human bacterial infections involve biofilms (Post et al., 2004). In recent years, the ability of GBS to form biofilm attracted attention for its possible role in fitness and/or virulence. Here, a new in vitro biofilm formation protocol was developed to guarantee more stringent conditions, to better discriminate between strong-, low- and non- biofilm forming strains and reduce ambiguous data interpretation. This protocol was applied to screen the in vitro biofilm formation ability of more than 350 GBS clinical isolates from pregnant women and neonatal infections belonging to different serotype, in relation to media composition and pH. The results showed the enhancement of GBS biofilm formation in acidic condition and identified a subset of isolates belonging to serotypes III and V that forms strong biofilms in these conditions. Interestingly, the best biofilm formers belonged to the serotype III hypervirulent clone ST-17.It was also found that pH 5.0 induces down-regulation of the capsule but that this reduction is not enough by itself to ensure biofilm formation. Moreover, the ability of proteinase K to strongly inhibit biofilm formation and to disaggregate mature biofilms suggested that proteins play an essential role in promoting GBS biofilm formation and contribute to the biofilm structural stability. Finally, a set of proteins potentially expressed during the GBS in vitro biofilm formation were identified by mass spectrometry.

la costa atlántica de España. Esta patología se caracteriza por la presencia de protuberancias que se evidencian a través de la piel de los peces afectados. Estas lesiones se corresponden con nódulos en el tejido muscular, que muestran un aspecto de absceso. Lesiones similares a éstas también se han detectado en riñón, corazón, hígado y tracto digestivo, lo que nos permite definir la enfermedad como sistémica. Las secciones histológicas de los nódulos revelaron un extenso núcleo compuesto mayoritariamente por tejido necrótico rodeado por fibroblastos y macrófagos. Además, organismos plasmodiales de morfología esférica se encontraron en la capa externa de estos nódulos, normalmente en el interior de macrófagos o fibroblastos. Estos organismos también se observaron en la mucosa y submucosa intestinal, sin causar lesiones aparentes. En este trabajo se ha podido identificar a estos organismos como una nueva especie de ameba perteneciente a la familia Entamoebidae (Phylum Amebozoa, Infraphylum Archamoeba), y se describe tentativamente como una nueva especie del género Endolimax, Endolimax piscium n. sp. E. piscium presenta trofozoitos redondeados (<5 μm) con un alto grado de simplificación intracelular, sin mitocondrias en el citoplasma pero con unos orgánulos compatibles con mitosomas. Con el fin de establecer técnicas fiables de diagnóstico para el reconocimiento de este parásito, se han desarrollado y evaluado técnicas específicas de hibridación in situ (ISH) y de reacción en cadena de la polimerasa (PCR), utilizando el examen histológico (una combinación de técnica histológica convencional en muestras de músculo e ISH en muestras de intestino) como prueba estándar para compararlos. Como resultado, todas las técnicas evaluadas obtuvieron altos indicadores de calidad. La técnica de ISH fue la más específica y sensible y se encontró particularmente útil como método de referencia de confirmación en muestras de intestino, no sólo para confirmar los positivos, sino también para descartar negativos, diagnosticados como dudosos por histología convencional. La técnica de PCR resulta ser un método rápido y fiable de diagnóstico, pero todavía necesita una mayor optimización metodológica de muestreo. Los resultados preliminares de la evaluación epidemiológica de la amebiasis en las diferentes granjas analizadas, sugieren que una vez la enfermedad se manifiesta en la granja, es muy probable que los peces asintomáticos también presenten parásitos en el intestino, aunque no necesariamente presenten lesiones. La ruta que estos organismos utilizan para atravesar la barrera intestinal, llegar a otros órganos y diseminarse sistémicamente en el interior del pez, causando graves lesiones, especialmente en el músculo, todavía no se conoce con exactitud. Sin embargo los estudios preliminares señalan al tejido conectivo como tejido diana, por lo que se cree que pueda tener un papel importante en esta difusión, aunque la vía hematógena no puede ser descartada. Además, se ha diseñado una infección experimental por cohabitación entre peces sanos y enfermos, mediante la cual se ha podido confirmar la transmisión horizontal del parásito, aunque ésta parece ser lenta y con un período prepatente largo. También se ha detectado E. piscium a partir de muestras de agua tomadas en el tanque donde se alojaban los peces enfermos, lo que apoya la hipótesis de que la transmisión pueda producirse e a través del agua. / A previously undescribed pathological condition is affecting the culture of Solea senegalensis in some farms of the Atlantic coast of Spain. This condition is characterised by the presence of external protuberances in the skin of the affected fish. These lesions correspond to nodules in the muscular tissue showing an abscess-like aspect. Similar lesions were found in kidney, heart, liver and the digestive tract, which leads us to define this pathology as a systemic disease. Histological sections of these nodules revealed the presence of a large core formed mainly of necrotic tissue surrounded with fibroblasts and macrophages. Round-shaped plasmodial organisms were found in the external layer of the nodules and usually inside macrophages or fibroblasts. These organisms were also observed in the intestinal mucosa and submucosa, without causing apparent lesions. This organisms are correspond to a new amoeba species, that belongs to the family Entamoebidae (Phylum Amebozoa, Infraphylum Archamoeba), and we tentatively describe it as a new species in the genus Endolimax, Endolimax piscium n. sp. E. piscium presents round to ovoid trophozoites (<5 μm) with a high degree of intracellular simplification. No mitochondria were observed but mitosome-like organelles were present. In order to establish reliable diagnostic techniques for the recognition of E. piscium, specific in Situ Hybridization (ISH) and Polymerase Chain Reaction (PCR) tests have been developed and evaluated using the histological examination (a combination of conventional histological technique in muscle samples and ISH in intestine samples) as gold standard to compare them. As a result, all evaluated techniques obtain quite high quality indicators. The ISH technique was the most specific and sensitive and it was useful as a reference confirmatory method in intestine samples, not only to confirm positives but also to discard negatives, diagnosed as doubtful by conventional histology. PCR technique is a fast and reliable routine method, but still needs further optimization of the sampling methodology. The preliminary results of epidemiological screening for the amoebiasis at the different farms suggest that once disease has manifested in a farm, it is quite probable that asymptomatic fish also present parasites within their intestine, although not necessarily presenting lesions. The route that these organisms use to breach through the intestinal barrier to infect other organs and spread systemically throughout the fish, causing serious lesions especially in muscle is still not completely understood. However preliminary studies point out the connective tissue as a preferential site where the parasites are observed so it would have some role of this dissemination, although haematogenous route of dissemination should not be discard. In addition, an experimental infection by cohabitation between healthy and diseased fish was designed whereby horizontal transmission of the parasite was confirmed, although it appears to be slow and with a long prepatent period. E. piscium stages were also detected from water samples from the cohabitating tank, which supports the hypothesis that transmission occurs through water.

La retinopatía diabética es la causa más común de ceguera adquirida en los países desarrollados, con una alta prevalencia en los pacientes diabéticos. El desarrollo de nuevas terapias requiere un buen conocimiento de la patología de la enfermedad y para ello son necesarios buenos modelos animales. Dichos modelos también resultan esenciales para ensayar el potencial de las nuevas terapias. El ratón transgénic IGF-I constituye un excelente modelo de retinopatía, ya que desarrolla la mayoría de las alteraciones vasculares presentes en los pacientes diabéticos. Para completar la caracterización del fenotipo de la retina de los animales TgIGF-I, la primera parte de esta tesis se centra en el estudio de las alteraciones de las neuronas y las células de la glia en las retinas transgénicas. Los resultados obtenidos muestran que los animales transgénicos presentan una pérdida progresiva de sus respuestas electroretinográficas resultando en una disfunción neuronal grave en los animales transgénicos de avanzada edad. Se detecta gliosis y microgliosis en las retinas de animales transgénicos jóvenes. La gliosis está relacionada con la pérdida de funciones esenciales para la supervivencia de las neuronas que realizan las células de Müller. Las retinas transgénicas presentan cambios en su metabolismo, relacionados con el reciclaje del glutamato, signos de estrés oxidativo y alteraciones en la homeostasis del potasio. Estas alteraciones pueden ser la causa de la disfunción neuronal observada en las retinas transgénicas, que además puede ser agravada por el incremento en la producción de citoquinas pro-inflamatorias. La segunda parte este trabajo se dedicó al estudio de la eficacia de una aproximación de terapia génica dirigida a contrarrestar la neovascularización y la neurodegeneración en la retinopatía diabética. Los vectores adeno-asociados (AAV) de tipo 2 fueron escogidos para sobreexpresar el Pigmented Epithelium Derived Factor (PEDF), una proteina con potentes propiedades antiangiogénicas y neuroprotectoras. La transferencia génica del PEDF mediante vectores AAV induce la expresión a largo plazo de esta proteína y, como consecuencia, una marcada reducción de la neovascularization intravítrea, la normalización de la densidad capilar de la retina y la prevención del desprendimiento de retina. Esta reversión del fenotipo es paralela a la reducción de los niveles intraoculares de VEGF y la regulación negativa de efectores de la angiogénesis. Estos resultados demuestran la eficacia a largo plazo del a sobreexpresión de PEDF para contrarrestar la neovascularización retinal y ofrece evidencias para el uso de esta estrategia en el tratamiento de la retinopatía diabética y otras enfermedades proliferativas de la retina. / Diabetic retinopathy (DR) is the most common cause of acquired blindness in developed countries, with a high prevalence in diabetic patients. The development of new effective therapies requires further investigations on disease pathogenesis and good animal models are essential to this end and to assay the potential efficacy of new experimental therapies. The TgIGF-I mice is a good model of retinopathy, developing many of the retinal vascular alterations observed in human diabetic patients. To fully characterize the eye pathology of the TgIGF-I, the first part of this work was focused in the study of the alterations of neurons and glial cells in the retinas of these mice. We found that TgIGF-I retinas showed a progressive decline in their electroretinographic responses that resulted in significantly impaired neuronal functionality in old animals. Gliosis and microgliosis were also detected in transgenic retinas at early ages. Gliosis is associated with the loss of essential neuron-supportive functions performed by Müller cells. We found that transgenic retinas showed changes in normal retinal metabolism, such as alterations in the glutamate metabolism, signs of oxidative stress and impaired potassium buffering, that may underlie neuronal dysfunction in transgenic retinas, which could be exacerbated by the increased production of pro-inflammatory cytokines. Thus, the second part of this work was dedicated to the study of the efficacy of a gene therapy approach aimed at counteracting neovascularization and neurodegeneration. Adeno-associated (AAV) vectors of serotype 2 were chosen to overexpress Pigmented Epithelium Derived Factor (PEDF), a protein with potent antiangiogenic and neuroprotective properties. AAV2-mediated PEDF gene transfer led to long-term production of PEDF and to a striking inhibition of intravitreal neovascularization, normalization of retinal capillary density, and prevention of retinal detachment. This was parallel to a reduction in the intraocular levels of Vascular Endothelial Growth Factor (VEGF), that was consistent with a downregulation of downstream effectors of angiogenesis. These results demonstrate long-term efficacy of AAV-mediated PEDF overexpression in counteracting retinal neovascularization and provide evidence towards the use of this strategy to treat angiogenesis in DR and other chronic proliferative retinal disorders.

S’han catalogat 156 líquens i fongs liquenícoles, saxícoles calcícoles, de 43 localitats de Mallorca i Cabrera. Es presenta per a cada espècie una descripció elaborada a partir del material recol•lectat, i es relacionen les localitats on s’ha trobat cada tàxon. Es consideren 41 espècies com a nova aportació a la flora de les Illes Balears,10 per a Mallorca i 67 per a Cabrera. S’estudia el paper dels líquens i altres litobionts en el procés de meteorització de les roques calcàries, a partir de les anàlisi de les aigües d’escorriment superficial sobre tres parells de roques calcàries, estudiant: conductivitat, pH, alcalinitat, clorurs, sulfats, calci, magnesi, sodi, potassi i amoni. A partir de la informació aconseguida de les ACP, així com de les mesures de biomassa i de la composició mineralògica de la zona micrititzada, es demostra l’ambivalència dels líquens saxícoles com a destructors / protectors de les roques calcàries / Se han catalogado 156 líquenes y hongos liquenícolas, saxícolas calcícolas, de 43 localidades de Mallorca y Cabrera. Para cada especie se presenta una descripción elaborada a partir del material recolectado y se relacionan las localidades donde se ha encontrado cada taxón. Se consideran 41 especies como nueva aportación a la flora de las Islas Baleares, 10 para Mallorca y 67 para Cabrera. Se estudia el papel de los líquenes y otros litobiontes en el proceso de meteorización de las rocas calcàreas, a partir de la analítica de las aguas de escorrentia superficial sobre tres pares de rocas calcàreas, considerando: conductividad, pH, alcalinidad, cloruros, sulfatos, calcio, magnesio, sodio, potasio y amonio. A partir de la información obtenida de los ACP, asi como de las medidas de biomasa y de la composición mineralógica de la zona micritizada, se demuestra la ambivalencia de los líquenes saxícolas como destructores / protectores de las rocas calcáreas / A total of 156 lichens and lichenocolous fongi, saxicolous calcareous, collected from 43 sites of Majorca and Cabrera, have been cataloged. For each of the species, we present a description obtained from the collected data, and the sites where the taxons come from are listed. We present 41 species as a new contribution to the flora of the Balearic Islands: 10 for Majorca and 67 for Cabrera. From the analysis of surface runoff waters on three pairs of limestones, we also study the role of lichens and other litobionts in the process of limestone weathering, by considering the following items: conductivity, pH, alkalinity, chloridres, sulfates, calcium, magnesium, sodium, potassium and ammonium. From the information obtained from the ACP, as well as the measures of biomass and the mineralogical composition of the micritized area, we make evident the ambivalence of the saxicolous lichens acting as destructive / protective agents of the limestone

El consumo de licuado de soja está experimentado un notable incremento debido a su consideración de producto saludable. El licuado de soja, además de ser una alternativa a la leche de vaca, sobre todo para las personas que poseen alguna intolerancia a los productos derivado de la leche, tiene componentes bioactivos (flavonoides, vitamina E y poliaminas) que pueden contribuir a prevenir algunas dolencias crónicas prevalentes en la sociedad actual. En este estudio se planteó la utilización de una tecnología emergente, la ultra alta presión de homogenización (UHPH) para la obtención de licuado de soja. Esta tecnología no térmica consiste en la aplicación de presiones de hasta 400 MPa utilizando un sistema de homogenización, especialmente diseñado para producir un efecto conservador, al mismo tiempo que se mejora la estabilidad coloidal y se mantiene la calidad nutricional y sensorial. Con esta hipótesis de partida, la UHPH podría ser una tecnología alternativa a las comúnmente aplicadas a nivel industrial. Por ello, en el planteamiento de este trabajo se incluyó el estudio comparativo de la UHPH con los tratamientos térmicos de pasteurización y UHT. En la primera parte de esta tesis se llevaron a cabo diferentes tratamientos UHPH (200 y 300 MPa con temperaturas de entrada de 55, 65 y 75ºC) con la finalidad de seleccionar las condiciones óptimas para obtener productos de buena calidad, tanto de almacenamiento en refrigeración, como de larga duración de almacenamiento a temperatura ambiente. El estudio se realizó a dos niveles independientes. Por una parte se evaluaron parámetros característicos de la calidad química, coloidal, enzimática y microbiológica de licuados de consumo habitual, y por la otra, se realizó un estudio con licuados inoculados con diversas cepas microbianas para conocer su cinética de destrucción frente a tratamientos UHPH. De estos estudios se concluyó que los tratamientos a 300 MPa produjeron licuado de soja con muy buena estabilidad coloidal y química y que, aplicando una temperatura de entrada de 85ºC en combinación con dicha presión, se alcanzó una excelente reducción de las esporas bacterianas. La segunda parte del trabajo consistió en el estudio de la evolución durante el almacenamiento de los licuados UHPH tratados en las condiciones óptimas seleccionadas del estudio previo. De este modo, se obtuvieron tanto licuados frescos similares a los pasteurizados, como licuados de larga duración, similares a los tratados por UHT. Para lograr este propósito, se evaluaron una serie de aspectos, tales como microbiológicos, estabilidad coloidal, cambios de color, parámetros químicos y sensoriales que permitieron evaluar la calidad global de los productos de soja, así como su aceptación por los consumidores. Los licuados de soja fresco y de larga duración alcanzaron respectivamente 1 y 6 meses de caducidad con mejor calidad que aquellos tratados térmicamente. / Soymilk consumption is experiencing a noticeable increase due to it being considered as a healthy product. Soymilk has often been used as an alternative to dairy milk for people who have intolerance to dairy products. Nowadays, it is known for its important health benefits that can contribute to the reduction of chronic illness commonly prevalent in the modern style life. This is due, primarily, to characteristics of protein fraction and minor components rich in antioxidant activity (flavonoids, tocopherols and poliamines) taking into account the excellent nutritional profile of soymilk. This thesis project was focused on the application of an emerging technology, Ultra High Pressure Homogenization (UHPH), in the production of soy vegetable milk. This non-thermal technology consists of a high pressure machine capable of applying pressures of up to 400 MPa using a special homogenizing system designed to produce a conserving effect, improving the colloidal stability while maintaining good nutritional and sensory qualities. Considering this hypothesis, UHPH could be an alternative technology to those commonly applied in the food industries. For that, a comparative study of UHPH with thermal treatments (pasteurization and UHT) was carried out in this work. In the first part of this thesis, different UHPH conditions (200 and 300 MPa at 55, 65 and 75ºC of inlet temperature) were performed on soymilk in order to select optimal treatment conditions for producing a good quality product whether intended for refrigeration or long-term storage at room temperature. In this first step, two independent evaluations were performed. On one hand, quality parameters related to chemical, enzymatic, microbiological and colloidal characteristics were evaluated and on the other hand, an inoculation study with different strain spores was carried out in order to determine the inactivation kinetic of the UHPH treatment. Results indicated that treatments at 300 MPa were able to produce soymilk with high chemical and colloidal stability. It is also worth noting that an excellent reduction of bacterial spores was reached applying inlet temperature of 85ºC at the same pressure. The second part consisted in the shelf-life evaluation of soymilk treated by UHPH using the selected optimal conditions determined in the previous step. As a result, soymilk was obtained with similar characteristics to those produced by pasteurization and with extended shelf-life similar to those obtained by UHT treatments. To achieve this purpose, microbiological aspects, colloidal stability, color changes, chemical parameters and sensory quality were applied to evaluate the overall quality of soymilk and its acceptance by the consumers. Refrigerated soymilk and that produced for an extended shelf-life respectively reached 1 and 6 months of storage in good conditions for consumption and with better quality than those obtained by thermal treatments.

La inocuidad de los alimentos constituye una de las preocupaciones de la industria de alimentos ya que afecta directamente a la salud de los consumidores. El queso chihuahua es el queso madurado de mayor consumo en México, la maduración se realiza mediante la adición de microorganismos lácticos, principalmente del género Lactococcus y en algunos casos también Streptococcus thermophilus y Lactobacillus. Se ha demostrado que en alimentos madurados se pueden desarrollar aminas biógenas por la presencia de enzimas amino descarboxilasas de los microorganismos fermentadores. El consumo de histamina y tiramina pueden ser de riesgo para los consumidores, en especial para quienes presentan inhibición de la enzima mono amino oxidasa. El objetivo de este trabajo fue evaluar el desarrollo de histamina y tiramina en 8 quesos chihuahua elaborados con leche pasteurizada durante la vida de anaquel. Se inició con el análisis de la calidad microbiológica y físico-química de los quesos requerida por la normatividad vigente, se desarrollaron cultivos en medios selectivos para identificar bacterias del queso con enzimas histidina y tirosina descarboxilasa (HDC y TDC) utilizando un caldo sintético con histidina o tirosina añadido, este estudio se realizó en 4 momentos de la vida de anaquel y para identificar las bacterias ácido láctico (BAL) con enzimas descarboxilasas en las pruebas positivas, se utilizó el sistema bioquímico API 50 CH (Biomerieux). Al mismo tiempo, se cuantificó la presencia de histamina y tiramina por cromatografía de líquidos de alta resolución (HPLC) en tres diferentes momentos, al inicio y a la mitad de la vida de anaquel, y en la fecha de caducidad del producto. Por otra parte, de los quesos se realizó una extracción directa de ADN bacteriano, donde se desarrolló una metodología que incluye un proceso de eliminación de la grasa. Se analizó la presencia de genes que codifican para las enzimas histidina y tirosina descarboxilasa por PCR (reacción de la cadena de la polimerasa) utilizando oligonucleótidos previamente reportados para hdc y diseñando nuevos oligonucleótidos para tdc. Los resultados microbiológicos mostraron que los quesos cumplen con la normatividad mexicana para estos productos, la cuenta total de bacterias fue de 3,10 a 3,80 Log UFC/g, de coliformes de 2,6 a 3,4 Log UFC/g. No se detectaron bacterias patógenas como Salmonella, Listeria monocytogenes ni Staphylococcus aureus. Los estudios físico químicos muestran variaciones importantes entre las marcas en las concentraciones de sodio, proteína y grasa en algunos casos los valores encontrados no corresponden a lo declarado en las etiquetas. La detección de microorganismos con enzima HDC al final de la vida de anaquel fue en un 37,5% de las muestras y para microorganismos con la enzima TDC en un 75%. Se identificaron Lactobacillus pentosus con enzimas HDC y TDC en 3 productos y L. rhamnosus con enzima TDC en 2 productos. Al inicio del almacenamiento ninguno de los quesos presentaba histamina y solo el 37,5% contenían tiramina en concentraciones de 34 a 122 mg/Kg de queso, mientras que al final de la vida de anaquel en el 75% de los quesos se detectó tiramina (115 a 209 mg/Kg de queso) y en el 37% se encontró histamina (47 a 205 mg/Kg de queso). En 6 de los 8 quesos analizados (75%) se detectó el gen tdc, mientras que en solo 3 (37,5%) se logró detectar el gen hdc. Al asociar los resultados de HPLC al final del almacenamiento con la presencia de los genes tdc y hdc se observa una correlación significativa de 0,001 y 0,024 respectivamente. Es indispensable disminuir la formación de aminas biógenas en los quesos ya sea por pasteurización de la leche, selección de las cepas iniciadoras y/o con buenas prácticas higiénicas. / Food safety is a concern in the food industry since it directly affects the consumer health. Chihuahua cheese is the mature cheese of major consumption in Mexico; the maturation is realized by adding lactic microorganisms, especially Lactococcus and in some cases Streptococcus thermophiles and Lactobacillus. It has been demonstrated that mature food can develop biogenic amines because of the presence of amino descarboxilasas enzymes of the fermenter microorganisms. The consumption of histamine and tyramine can be risky for consumers, especially for those who present inhibition of monoamine oxidase enzyme. The aim of this study was to evaluate the development of histamine and tyramine in 8 chihuahua cheeses elaborated with pasteurized milk during its shelf life. It began with the analysis of the microbiological and physical-chemical quality of the cheeses required by current regulations; selective media cultures were developed to identify bacteria in cheeses with enzymes histidine and tyrosine decarboxylase (HDC y TDC) using a synthetic broth with histidine or tyrosine added, this study was made in 4 times of the shelf life and to identify the lactic acid bacteria (BAL) with descarboxilasa enzyme in the positive tests, the biochemical system API 50 CH (Biomerieux) was used. At the same time, the presence of histamine and tyramine was quantified by high- performance liquid chromatography (HPLC) in three different moments, at the beginning and middle of shelf life and at the product expiration. On the other hand, bacterial ADN was extracted directly from cheese to develop a methodology that includes a fat removal process. It was analyzed the presence of genes that encode the histidine and tyrosine decarboxylase enzyme by PCR reaction (Polymerase Chain) using primers previously reported for hdc and designing new primers for tdc. The microbiological results showed that the cheeses fulfill the Mexican regulations for these products, the total account of bacteria was from 3,10 to 3,80 Log UFC/g, and coliformes from 2,6 to 3,4 Log UFC/g. Pathogenic bacteria such as Salmonella, Listeria monocytogenes or Staphylococcus aureus were not found. The physical-.chemical studies show important variations between the marks in the concentrations of sodium, protein and fat, in some cases the values do not correspond to the ones declared in the labels. The detection of microorganisms with enzyme HDC at the end of shelf life was 37,5% of the samples and microorganisms with the enzyme TDC was 75%. Lactobacillus pentosus were identified with enzymes HDC and TDC in 3 products and L. rhamnosus with enzyme TDC in 2 products. At the beginning of storage none of the cheeses had histamine and only 37.5% contained tyramine in concentrations from 34 to 122 mg/Kg of cheese, whereas at the end of the shelf life 75% of the cheeses had tyramine (115 to 209 mg/Kg of cheese) and 37% histamine (47-205 mg/Kg of cheese). Six of the eight cheeses analyzed (75%) had tdc gene whereas only 3 (37,5%) contained the hdc gene. The results of HPLC at the end of storage in the presence of tdc and hdc genes show a significant correlation of 0,001 and 0,024 respectively. It is indispensable to decrease the formation of biogenic amines in cheeses either by pasteurization of milk, selection of the strains initiators and/or with good hygiene practices.

El sistema immunitari innat es basa en el reconeixement no específic d'elements conservats del metabolisme dels patògens. Aquest reconeixement es fa principalment a través de receptors de reconeixement de patrons (PRRs) codificats per la línia germinal, que són presents a cèl·lules especialitzades del sistema immunitari innat, i que són capaços de reconèixer patrons moleculars conservats associats a patògens (PAMPs). Aquest reconeixement iniciarà diferents vies de senyalització que induiran la transcripció de citoquines proinflamatòries per finalment donar lloc a una inflamació local. D'aquesta manera, el sistema immunitari innat pot ser modulat, a través de l'administració d'aquests PAMPs, simulant una trobada natural entre el sistema immunitari i els patògens. La principal hipòtesi d'aquest estudi va ser que, mitjançant l'encapsulació en un mateix sistema d'administració nanomètric de diversos PAMPs, també anomenats immunoestimulants, es podria millorar la seva administració a diferents espècies de peixos. També, que aquest sistema d'administració podria interaccionar amb les cèl·lules del sistema immunitari generant la seva activació no específica, i millorant la resposta immunitària contra diferents malalties infeccioses. En aquest context, s'ha desenvolupat un nou sistema d'administració d'immunoestimulants basat en liposomes que encapsulen el lipopolisacàrid bacterià d'Escherichia coli, i un anàleg sintètic de dsRNA viral, el poli (I:C). Els nostres resultat van mostrar que aquests liposomes eren biocompatibles i capaços de ser endocitats in vitro per hepatòcits de peix zebra (Danio rerio) i per macròfags de truita irisada (Oncorhynchus mykiss). Així mateix, els liposomes van poder modular in vitro l'expressió de diversos gens relacionats amb la immunitat. També s'ha desenvolupat un mètode per a la captació d'imatges in vivo dels liposomes nanomètrics en adults de peix zebra. Això ens va permetre seguir la dinàmica i els teixits diana dels liposomes administrats tant per injecció intraperitoneal com per immersió. Els resultats dels estudis de biodistribució van demostrar que els liposomes s'acumulaven principalment a la melsa del peix zebra i en cèl·lules del sistema immunitari com ara macròfags de truita irisada. D'altra banda, hem demostrat que aquests liposomes, administrats mitjançant injecció intraperitoneal i immersió, podrien protegir de manera efectiva el peix zebra tant d'una infecció bacteriana (Pseudomonas aeruginosa PAO1) com viral (virèmia primaveral de carpa). En conclusió, els resultats suggereixen que l'estimulació del sistema immunitari innat amb liposomes que encapsulen un lipopolisacàrid bacterià i l'anàleg sintètic de dsRNA viral, poli (I:C), podria ser una bona estratègia per aconseguir la protecció contra infeccions bacterianes i virals, i que per tant, es podria utilitzar potencialment com una eina no específica per a la prevenció d'infeccions en peix. / The innate immune system is based on the non-specific recognition of conserved elements of the pathogenic metabolism. This recognition is primarily mediated by germ line encoded pattern-recognition receptors (PRRs), present in specialized cells of the innate immune system, that are able to recognize conserved molecular patterns associated to pathogens (PAMPs). This recognition will trigger different signaling pathways that will induce the transcription of pro-inflammatory cytokines and result in local inflammation. Therefore, the innate immune system can be modulated by administration of these PAMPs, simulating a natural pathogen–immune system encounter. The main hypothesis of this study was that, by encapsulation in the same nanoscaled delivery system, of several PAMPs, also called immunostimulants, we could improve their administration to different fish species. Also, that this delivery system would interact with the cells of the immune system generating its non-specific activation and improving the immune response against different infectious diseases. In this context, a novel immunostimulant delivery nanosystem based on liposomes encapsulating a bacterial lipopolysaccharide from Escherichia coli, and a synthetic analog of viral dsRNA, poly (I:C), has been developed. Our data shows that, these biocompatible liposomes were able to be endocytosed in vitro by zebrafish (Danio rerio) hepatocytes and rainbow trout (Oncorhynchus mykiss) macrophages as well as to regulate the expression of immune related genes. We have also developed a method for in vivo imaging of nano-sized liposomes in adult zebrafish, which allowed us to follow the dynamics and the target tissues of the liposomes administered either by intraperitoneal injection or immersion. The biodistribution results showed that the delivery system accumulated mainly in the spleen of zebrafish and in immune relevant cells, such as macrophages, from rainbow trout. Moreover, we showed that these liposomes, administrated by intraperitoneal injection and immersion, could effectively protect zebrafish from bacterial (Pseudomonas aeruginosa PAO1) and viral (spring viraemia of carp virus) infections. In conclusion, these findings suggest that the stimulation of the innate immune system with liposomes encapsulating a bacterial lipopolysaccharide and the synthetic analog of viral dsRNA, poly (I:C), could be a good strategy to achieve protection against bacterial and viral infections therefore potentially working as a non-specific prevention tool in fish.

From the late 1980s, the automation of sequencing techniques and the computer spread gave rise to a flourishing number of new molecular structures and sequences and to proliferation of new databases in which to store them. Here are presented three computational approaches able to analyse the massive amount of publicly avalilable data in order to answer to important biological questions. The first strategy studies the incorrect assignment of the first AUG codon in a messenger RNA (mRNA), due to the incomplete determination of its 5' end sequence. An extension of the mRNA 5' coding region was identified in 477 in human loci, out of all human known mRNAs analysed, using an automated expressed sequence tag (EST)-based approach. Proof-of-concept confirmation was obtained by in vitro cloning and sequencing for GNB2L1, QARS and TDP2 and the consequences for the functional studies are discussed. The second approach analyses the codon bias, the phenomenon in which distinct synonymous codons are used with different frequencies, and, following integration with a gene expression profile, estimates the total number of codons present across all the expressed mRNAs (named here "codonome value") in a given biological condition. Systematic analyses across different pathological and normal human tissues and multiple species shows a surprisingly tight correlation between the codon bias and the codonome bias. The third approach is useful to studies the expression of human autism spectrum disorder (ASD) implicated genes. ASD implicated genes sharing microRNA response elements (MREs) for the same microRNA are co-expressed in brain samples from healthy and ASD affected individuals. The different expression of a recently identified long non coding RNA which have four MREs for the same microRNA could disrupt the equilibrium in this network, but further analyses and experiments are needed.

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by genetically diverse Neisseria meningitidis strains and is an antigen of the multicomponent protein-based 4CMenB vaccine, able to induce bactericidal antibodies in humans and to bind heparin-like molecules. The aim of this study is to characterize the immunological and functional properties of NHBA. To evaluate immunogenicity and the contribution of aminoacid sequence variability to vaccine coverage, we constructed recombinant isogenic strains that are susceptible to bactericidal killing only by anti-NHBA antibodies and engineered them to express equal levels of selected NHBA peptides. In these recombinant strains, we observed different titres associated with the different peptide variants. These recombinant strains were then further engineered to express NHBA chimeric proteins to investigate the regions important for immunogenicity. In natural strains, anti-NHBA antibodies were found to be cross-protective against strains expressing different peptides. To investigate the functional properties of this antigen, the recombinant purified NHBA protein was tested in in vitro binding studies and was found to be able to bind epithelial cells. The binding was abolished when cells were treated specifically with heparinase III, suggesting that the interaction with the cells is mediated by heparan sulfate proteoglycans (HSPG). Mutation of the Arg-rich tract of NHBA abrogated the binding, confirming the importance of this region in mediating the binding to heparin-like molecules. In a panel of N. meningitidis strains, the deletion of nhba resulted in a reduction of adhesion with respect to each isogenic wild type strain. Furthermore, the adhesion of the wild-type strain was prevented by using anti-NHBA polyclonal sera, demonstrating the specificity of the interaction. These results suggest that NHBA could be a novel meningococcal adhesin contributing to host-cell interaction. Moreover, we analysed NHBA NalP-mediated cleavage in different NHBA peptides and showed that not all NHBA peptides are cleaved.