Nitrogen availability and transformation in acidified and N saturated soils of Czech (The Bohemian Forest, Ore Mountains) and Ukraine (Pop Ivan massif) mountain forest ecosystems were investigated. The study was primarily focused on the role of microbial immobilization in soil N retention. The study was based on field measurements (ion exchange resins), analyses of selected soil biochemical and microbial characteristics, and on laboratory experiments (15N labelling).

Thesis (PhD (Chemistry and Polymer Science))--Stellenbosch University, 2008. / The successful preparation and structural characterization of a number of N,N-dialkyl-N’-benzoyl(thio)selenourea ligands is described; where the intermolecular interactions are characterized by the presence of Resonance Assisted Hydrogen Bonding (RAHB), π- π interactions between neighbouring benzene residues only being evident amongst the longer alkyl chain derivatives. The first structural characterization of an asymmetrically substituted N,N-dialkyl- N’-benzoylselenourea ligand reveals an increased stability of the Z isomer in the solid state, this being reflected by the sulfur analogue. Attempts to synthesise N,N-dicyclohexyl-N’-benzoylselenourea led to the isolation and structural characterization of a novel 1,3,5-oxaselenazine salt and dicyclohexylaminobenzoate. The first structural characterization of a “bipodal” N,N-dialkyl-N’-benzoylselenourea ligand, 3,3,3’,3’-tetrabutyl-1,1’- isophthaloylbis(selenourea), reveals RAHB in the crystal lattice similar to that exhibited by the “monopodal” analogue, N,N-dibutyl-N’-benzoylselenourea. The successful complexation of the N,N-dialkyl-N’-benzoyl(thio)selenourea ligands to a number of different transition metal ions is reported allowing the preparation of several potential single source precursors. Coordination through the O and Se/S donor atoms to Pd(II) results in the formation of square planar metal complexes, with a cis conformation, several of which could be structurally characterized. In particular, the first structural elucidation of an asymmetrically substituted N,N-dialkyl-N’-benzoylselenourea metal complex, cis-bis(N-benzyl-N-methyl-N’- benzoylselenoureato)palladium(II) indicates the increased stability of the EZ isomer in the solid state. Structural elucidation of the novel (N,N-diphenyl-N’-benzoylselenoureato)cadmium(II) reveals a bimetallic complex in the solid state, where the expected 2:1 ligand : metal ratio is maintained, and the two Cd(II) centres are 5 and 6 coordinated, with O and Se donor atoms. Multinuclear Nuclear Magnetic Resonance (NMR) Spectroscopy has been employed in the thorough characterisation of the potential single source precursors, 77Se NMR spectroscopy indicating a decreased shielding of the 77Se nucleus as the “hardness” of the central metal ion increases i.e. Pd(II) > Zn(II) > Cd(II). Use of 113Cd NMR spectroscopy indicates the preferential binding of N,N-diethyl-N’- benzoylselenourea to Cd(II) over that of its sulfur analogue, and initial studies suggest a form of chelate metathesis taking place in solution. 31P NMR spectroscopy is used to gain insight into the formation of cis-bis(N,N-diethyl-N’- benzoylselenoureato)Pt(II). Thermolysis of (N,N-diethyl-N’-benzoylselenoureato)cadmium(II) and its sulfur analogue led to the successful synthesis of CdSe and CdS quantum dots respectively, where thermolysis over a range of temperatures allows a degree of size control over the resulting nanoparticles. The effect of precursor alkyl chain length on nanoparticle morphology was investigated for both the N,N-dialkyl-N’-benzoylthio- and –selenoureas. A correlation between the two for the (N,N-dialkyl-N’-benzoylselenoureato)Cd(II) complexes is described and possible growth mechanisms are discussed. Preliminary investigations into the use of other N,N-dialkyl-N’-benzoyl(thio)selenourea metal complexes as single source precursors reveal that both (N,N-diethyl-N’-benzoylselenoureato)Zn(II) and its sulfur analogue show potential as single source precursors for the formation of ZnO and ZnS nanoparticles respectively. Initial studies into the use of N,N-dialkyl-N’-benzoyl(thio)selenourea metal complexes as single source precursors for the synthesis of core-shell nanoparticles is briefly described. The Aerosol Assisted Chemical Vapour Deposition (AACVD) of several N,N-dialkyl-N’-benzoyl(thio)selenourea metal complexes is reported, where both (N,N-diethyl-N’-benzoylselenoureato)Cd(II) and its sulfur analogue allow the deposition of crystalline CdSe and CdS respectively. The AACVD of (N,N-diethyl-N’- benzoylselenoureato)Zn(II) leads to the deposition of crystalline ZnSe, ZnS being deposited by (N,N-diethyl-N’-benzoylthioureato)Zn(II). The deposition of heazelwoodite (Ni3S2) with varying morphologies results from the AACVD of cis-bis(N,N-diethyl-N’-benzoylthioureato)Ni(II). Thermal annealing of the amorphous material deposited by the AACVD of cis-bis(N,N-diethyl-N’-benzoylthioureato)Pd(II), allows the formation of highly crystalline palladium. The deposition of metallic platinum using cis-bis(N,N-diethyl-N’-benzoylthioureato)Pt(II) is described as well as the deposition of crystalline Pd17Se15 from cis-bis(N,N-diethyl-N’-benzoylselenoureato)Pd(II). This, to the best of our knowledge, is the first time that AACVD has been performed, using the N,N-dialkyl-N’- benzoyl(thio)selenourea metal complexes as single source precursors, in addition, we believe it to be the first time that palladium selenide has been deposited using the AACVD technique.

Tesis para optar al grado de Mag?ster en Gesti?n para la Globalizaci?n / El objetivo de este trabajo es presentar un an?lisis de mercado y plan de negocios de la empresa nutricional espa?ola Nutrici?n 3G, el cual es un emprendimiento de investigadores espa?oles con amplia experiencia en el ?mbito universitario. Su servicio es vendido a los profesionales nutricionistas, quienes se los ofrecen a sus pacientes. Se toma una muestra de saliva de los pacientes y por medio de un an?lisis realizado en la casa matriz en Espa?a, entregan las probabilidades de desarrollar diversas enfermedades no transmisibles, tales como diabetes, dislipidemias, osteoporosis, ictus, etc. En Espa?a, Nutrici?n 3G ha tenido relativo ?xito y est? en condiciones financieras y de recursos de buscar nuevos clientes en otros mercados, siendo Latinoam?rica, la alternativa m?s factible. Se realiz? un an?lisis de cuatro mercados potenciales; Chile, Per?, Colombia y M?xico, resultando Chile el pa?s con mayor potencial de entrada y penetraci?n del servicio por medio de un representante en el pa?s. El mercado presenta una gran cantidad de competidores directos e indirectos y un p?blico objetivo muy espec?fico (profesionales nutricionistas) sin embargo, el an?lisis del negocio, utilizando una tasa de descuento de 10,24,%; arroj? una recuperaci?n de la inversi?n inicial al cabo de dos a?os, una tasa interna de retorno de 48% y VAN a los 5 a?os de 5.479 Euros. Situaci?n similar se dan en el an?lisis de escenario. Se evalu? la variaci?n en la venta de kits con variaciones del 10% (+10% en el caso optimista y -10% en el caso pesimista) presentando ambos casos recuperaci?n de la inversi?n de aproximadamente 2 a?os; TIR?s de 50% y 33%; ambas superiores a la tasa de descuento y con VAN?s positivas. Como conclusi?n, pese a lo competitivo del mercado, se recomienda el lanzamiento de Nutrici?n 3G en el mercado chileno.

Degradation of peroral administered drugs by various enzymes in the gastrointestinal tract has proven to be troublesome for the absorption' and bioavailability of protein and peptide drugs. Mucoadhesive polymers such as poly(acrylates) have proven to inhibit protease enzymes responsible for initiating digestion of peptide drugs. Enzyme inhibitors have unique chemical properties enabling it to interact with enzymes to form complexes with such enzymes prohibiting it from functioning properly. Anionic carboxymethylated chitosan derivatives such as N,N-dicarboxymethyl chitosan and N, O-carboxymethyl chitosan display unique structural similarities to enzyme inhibitors being anionic polymers that may interact with bi-valent cations... / Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.

The cytosolic protein degradation pathway, performed by ATP-dependent proteases and ATP-independent peptidases, plays important roles in several cellular activities, e.g. cell division, cell cycle progression, intracellular signaling, MHC class I antigen presentation, host-pathogen interactions, etc. The roles of ATP-dependent proteases during stress and infection have been studied in great detail but the functional roles of ATP-independent peptidases are not clearly understood. In this study, the functional roles of E. coli or S. typhimurium encoded Peptidase N (PepN), an ATP-independent enzyme belonging to theM1 family of metallopeptidases, were investigated. The thesis will address four different aspects. (i) In the first part, the utility of using E coli ∆pepN to identify and characterize novel peptidases will be shown. It is known that deletion of pepN leads to inability to cleave the majority of in vitro peptidase substrates in E. coli and S. typhimurium. To study the differences between two closely related paralogs of the M17 family, E. coli encoded pepA and pepB were cloned in pBAD24 vector and introduced in E. coli ∆pepN. Peptidase A (PepA) and Peptidase B (PepB) expression increases the cleavage of several aminopeptidase substrates and partially rescues growth of ∆pepN during nutritional downshift and high temperature stress (NDHT), a dual stress involving growth in minimal media at 42°C. Purified PepA and PepB enzymes display broad substrate specificity; however, distinct differences are observed between these two paralogs: PepA is more stable at high temperature whereas PepB displays broader substrate specificity as it cleaves Asp and Insulin B chain peptide. The strategy utilized in this study, i.e. overexpression of peptidases in ∆pepN followed by screening for substrate specificities in total cell extracts, may be used to rapidly identify the substrate preferences of novel peptidases encoded in genomes of different organisms. (ii) The second aspect investigates the functional roles of PepN during stress and infection in S. typhimurium. PepN has two conserved signature motifs of the M1 family, GAMEN and HEXXH, which play roles in substrate recognition and catalysis. To address the roles of catalytic activity of PepN, the residue E-298, which is present in the HEXXH motif and acts as a general base during catalysis, was mutated to A-298 by site-specific mutagenesis and introduced into ∆pepN (pBR322/pepNE298A). Biochemical and biophysical analysis of purified PepN (WT and E298A) revealed loss of catalytic activity of E298A but no major structural changes were observed in comparison to the WT protein. The functional roles of this mutation using ∆pepN expressing pBR322/pepN or pBR322/pepNE298A were investigated using two conditions: (i) Nutritional downshift high temperature (NDHT)stress and (ii) systemic infection in mice. Monitoring growth profiles of different strains demonstrated the requirement of the enzymatic activity of PepN for adaptation and growth to NDHT stress. Earlier studies have shown that S. typhimurium ∆pepN hyper proliferates in peripheral organs during systemic infection in mice. However, expression of wild type (WT)or E298A PepN led to lower colony forming units (CFU), demonstrating that the decrease in CFU is independent of catalytic activity. These observations are consistent with lower serum amounts of inflammatory cytokines, lower tissue damage and increase in survival of mice infected with S. typhimurium expressing WT or E298A PepN. (iii) Although pathogen encoded peptidases are known to be important during infection, their roles in modulating host responses in immunocompromised individuals are not well studied. In the third part of this thesis, the roles of S. typhimurium encoded PepN were studied in mice lacking Interferon-γ (Ifnγ), a cytokine important for immunity. S. typhimurium lacking pepN displays enhanced CFU compared to WT in peripheral organs during systemic infection in C57BL/6 mice. However, Ifnγ-/-mice show higher CFU compared to C57BL/6 mice, resulting in lower fold differences between WT and ∆pepN. Concomitantly, reintroduction of pepN in ∆pepN reduces CFU, demonstrating pepN dependence. In addition, three distinct differences were observed between infection ofC57BL/6 and Ifnγ-/-mice upon infection with different S. typhimurium strains: (i) cytokine profiles, (ii) histological analysis and (iii) mice survival. Overall, the roles of the host encoded Ifnγ during infection with S. typhimurium strains with varying degrees of virulence will be highlighted. (iv) The final aspect of this study reveals differences in gene expression between S. typhimurium grown in rich medium (Luria-Bertani) versus NDHT stress. This adaptation affects several pathways and the gene expression of secretory proteins that are important for virulence in S. typhimurium are greatly reduced during NDHT stress. Also, analysis of secretory protein amounts in different media conditions shows reduction during growth in minimal media plus high temperature stress. The functional consequences of this reduction in secretory protein amounts lead to lower bacterial replication after infection of RAW cells or mice infected via the oral route. In addition, the differences in gene expression between WT and ∆pepN during these conditions were studied. Interestingly, there is reduction in expression of flagellar genes whereas the genes involved in nitrogen metabolism are upregulated in ∆pepN upon exposure to NDHT stress. Further studies were performed by quantifying the motility of different S. typhimurium strains grown in a variety of culture conditions. Overall, this part of the study attempts to compare and contrast the possible adaptive responses of WT and ∆pepN to NDHT stress. Together, this thesis addresses multiple aspects of the biochemistry and roles of the enigmatic PepN during stress and infection.

Degradation of peroral administered drugs by various enzymes in the gastrointestinal tract has proven to be troublesome for the absorption' and bioavailability of protein and peptide drugs. Mucoadhesive polymers such as poly(acrylates) have proven to inhibit protease enzymes responsible for initiating digestion of peptide drugs. Enzyme inhibitors have unique chemical properties enabling it to interact with enzymes to form complexes with such enzymes prohibiting it from functioning properly. Anionic carboxymethylated chitosan derivatives such as N,N-dicarboxymethyl chitosan and N, O-carboxymethyl chitosan display unique structural similarities to enzyme inhibitors being anionic polymers that may interact with bi-valent cations... / Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.

La position a priori anti-ottomane, élaborée par des philosophes comme Volney et Montesquieu, en Syrie a été largement partagée par les voyageurs du XIXe siècle et même complétée par l'ensemble des travaux réalisés tout au long du XIXe siècle. Ainsi, voyageurs scientifiques, philosophes, consuls, journalistes ou même poètes, se sont presque tous impliqués dans l'élaboration et la circulation des clichés et des idées reçues sur la Syrie et sur le système turc en Syrie perçu comme une tyrannie globale. Les voyageurs participent ainsi avec leurs écrits, directement ou indirectement, à pérenniser la vision du « despotisme oriental» élaborée par leurs prédécesseurs et relayée dans un premier temps par Volney. Celui-ci, à peine arrivé en Syrie en 1783, dénonçait la misère dont il rendait aussitôt responsables les Turcs. Les autres voyageurs ont presque tous fait le concept de despotisme oriental et ont approuvé dans leurs récits l'auteur du Voyage en Égypte et en Syrie. D'une manière générale, on comprend que l'espace syrien évoque aux voyageurs des souvenirs ancestraux et leur rappelle un passé aussi bien religieux qu'historique parfois très proche. Par là-même, la Syrie, exotique, mythique ou pittoresque, a représenté un intérêt tout particulier pour les voyageurs-écrivains, les touristes et les scientifiques en quête d'exotisme et d'un retour aux sources. / The a priori anti-Ottoman position, developed by philosophers such as Volney and Montesquieu, in Syria, was largely shared by travelers in the 19th century. This preconceived philosophy was reinforced and "completed" by the entire collection of works written throughout the 19th century. Traveling scientists, philosophers, consuls, journalists, and even poets were almost all involved in developing and circulating ideas and clichés regarding Syria and the idea that Turkish rule in Syria was tyrannous. Travelers, as well as their writings, directly or indirectly perpetuated the vision of "Oriental tyranny." This vision, developed by predecessors, was first communicated in writing by Volney. In 1783, he had barely just arrived in Syria, when he began denouncing the misery he encountered, forwhich he held Turkish officials immediately responsible. Other travelers almost all supported the idea of "Oriental Tyranny" and agreed with the author of Voyage en Egypte et en Syrie in their writings.Generally speaking, Syria makes travelers think of their forefathers and reminds them of the past (in both a historic and religious context) that is in reality not that distant from the present. Moreover, those in search of getting back to the basics and the exotic; traveling writers, tourists, and scientists, took a particular interest in the exotic, mythic or picturesque Syria.

Les hétérocycles azotés occupent une place importante en chimie organique et médicinale et, à ce titre, constituent des cibles de choix pour le développement de réactions toujours plus efficaces et faciles d’emploi. Les travaux antérieurs, réalisés au laboratoire URCOM sur l’accès aux γ-lactames, ont servi de base pour développer une méthodologie impliquant des conditions douces qui ont mis en évidence les propriétés spécifiques des N-alkoxy amides pour accéder aux γ- et δ-lactames hautement fonctionnalisés de manière hautement diastéréosélective selon un processus domino. Cette méthodologie a aussi pu être étendue aux hydantoïnes, 2-imino-thiazolidin-4-ones et thiazinan-4-ones qui constituent également des molécules étudiées en chimie médicinale. En parallèle, une comparaison de réactivité entre N-alkoxy amides et N-alkyl amides a montré la meilleure réactivité des composés N-alkoxylés pour l’obtention de ces différents hétérocycles azotés. Cette différence de réactivité a été attribuée à la possible chélation du contre anion alcalins des bases minérales utilisées dans notre approche par les N-alkoxy amides. Les conditions douces ont permis d’envisager des versions organocatalysées énantiosélectives ou multicomposantes des réactions précédentes conduisant à des résultats préliminaires intéressants. / Nitrogen-containing heterocycles are key compounds in organic and medicinal chemistry and continue to attract synthetic effort for the development of more efficient and convenient reaction. Previous work carried out at the URCOM laboratory for the access to γ-lactams has provided the basis for developing a highly diastereoselective methodology under mild conditions that has demonstrated the specific properties of N-alkoxy amides, allowing the access to γ- and δ-lactams following a domino process. This methodology also allowed the access to hydantoïns, 2-imino thiazolidin-4-ones and thiazinan-4-ones which are compounds of importance in medicinal chemistry. In parallel, a comparison of reactivity between N-alkoxy and N-alkyl amides was carried out in order to demonstrate the better reactivity of the N-alkoxy amide compounds. This better reactivity was attributed to a possible chelation of the alkali metal counter anion of the mineral bases used in our conditions by the N-alkoxy amides. These mild conditions allowed organocatalyzed enantioselective or multicomponents approaches providing interesting preliminary results.

[EN] Human milk contains a large number of oligosaccharides, either free or bound to proteins and lipids, and their physiological role is mostly unknown. These oligosaccharides are resistant to host gastrointestinal digestion and therefore, a significant proportion reaches the infant colon, where they can be substrates for the resident microbiota. Lacto-N-biose (LNB) and galacto-N-biose (GNB) are the core type-1 sugar structures in HMO and mucin glycoproteins, respectively. They are fermented by species of the genus Bifidobacterium, but, there is no data about their utilization by the genus Lactobacillus. There is also no information for this genus about the metabolism of N-acetyllactosamine (LacNAc), which constitutes the type-2 sugar core in HMO, and lacto-N-triose, which forms part of the structure of lacto-N-tetraose, one of the most abundant HMOs. Lactobacillus casei is a lactic acid bacteria isolated from several environmental niches such as milk, meat, and reproductive and gastrointestinal tracts of animals and humans. In addition, some strains are used as starter cultures in the dairy industry and also as probiotics. The capability of L. casei species to survive in the gastrointestinal tract would depend in part of its ability to metabolize the available carbohydrates. In this Thesis we have shown that this strain is able to grow using LNB, GBN, LacNAc, and lacto-N-triose as carbon sources, and we have characterized the corresponding metabolic pathways. L. casei contains a gene cluster, gnbREFGBCDA, involved in the metabolism of GNB, LNB and also N-acetylgalactosamine. Transcriptional analysis showed that the gnb operon is regulated by substrate-specific induction mediated by the transcriptional repressor GnbR. Upstream of the gnb operon, there are two genes, bnaG and manA, encoding a b-N-acetylglucosaminidase precursor and a mannose-6P isomerase. It has been shown that BnaG is an extracellular wall-attached enzyme and that it is involved on lacto-N-triose metabolism. ManA enzyme is involved in the utilization of the mannose moiety of 3'-N-acetylglucosaminyl-mannose, which is a carbon source for L. casei BL23. Finally, in this strain, LacNAc is transported and phosphorylated by the lactose PTS and it is intracellularly hydrolyzed by the phospho-b-galactosidase LacG into galactose-6P and GlcNAc. Transcriptional analysis showed that the lac operon, in addition to lactose, is also induced by LacNAc. In an effort to better understand the metabolism and bioactive potential of HMO, sufficient quantities are required. In order to have enough amounts of LNB and GNB to test their biological activities, both disaccharides have been synthesized in vitro using the transglycosylation activity of the GnbG glycosyl hydrolase isolated from L. casei. Transglycosylation reactions were scaled and the resulting products were purified, and the yields obtained were 10.7 ± 0.2 g/L of LNB and 10.8 ± 0.3 g/l of GNB. Both disaccharides were used in vitro to determine their potential prebiotic properties using 33 Lactobacillus strains corresponding to 13 different species. It was determined that 21 strains, corresponding to the species L. casei, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus gasseri and Lactobacillus johnsonii were able to metabolize both GNB as LNB. Recently, some scientific evidences suggest an immunomodulatory function for HMO. In vitro analyses to assay this function have been performed for LNB and GNB, and for two fucosyloligosaccharides (Fuc-a-1,3-GlcNAc and Fuc-a-1,6-GlcNAc) previously synthesized in our laboratory. The four disaccharides were able to significantly increase IFN-g production in Peripheral Blood Mononuclear Cells (PBMC). Fuc-a-1,6-GlcNAc was also able to significantly reduce the production of IL13. These results suggest a stimulatory effect on the immune system, and in the particular case of Fuc-a-1,6-GlcNAc, a polarizing effect of immune response Th1 / Th2 towards Th1 populations. / [ES] La leche humana contiene una gran cantidad de oligosacáridos, libres o unidos a lípidos y proteínas, y su función fisiológica es mayoritariamente desconocida. Estos oligosacáridos son resistentes a la digestión y una gran proporción llega al intestino del lactante, donde pueden ser substratos para la microbiota. La lacto-N-biosa (LNB) y la galacto-N-biosa (GNB) son las estructuras tipo-1 que forman el núcleo de los oligosacáridos de la leche humana (OLH) y de las glicoproteínas de la mucina, respectivamente. Los dos azúcares son fermentados por especies del género Bifidobacterium, pero no hay datos acerca de su utilización por el género Lactobacillus. Tampoco hay información para este género acerca del metabolismo de la N-acetil-lactosamina (LacNAc), que constituye la cadena de azúcar tipo-2 en los OLH, y de la lacto-N-triosa, que forma parte de la estructura de la lacto-N-tetraosa, uno de los OLH más abundantes. La capacidad de Lactobacillus casei, de prevalecer en el sistema gastrointestinal dependerá en parte de su versatilidad metabólica para utilizar los carbohidratos disponibles. En la presente Tesis Doctoral se ha demostrado que la cepa L. casei BL23 se puede cultivar en presencia de LNB, GBN, LacNAc, y lacto-N-triosa como fuentes de carbono. En el metabolismo de la LNB, GNB y también N-acetilgalactosamina (GalNAc) está implicado el operon gnbREFGBCDA. Análisis transcripcionales demostraron que el operon gnb está regulado por inducción del substrato mediada por el represor transcripcional GnbR. Por encima del operon gnb, hay dos genes bnaG y manA, que codifican para el precursor de una b-N-acetilglucosaminidasa y una manosa-6P isomerasa. Se ha demostrado que BnaG es un enzima extracelular unido a la pared celular y que está implicado en el metabolismo de la lacto-N-triosa. El enzima ManA está implicada en el metabolismo de la manosa presente en el disacárido 3'-N-acetilglucosaminil-manosa, el cual es también una fuente de carbono para L. casei BL23. Por último, en esta cepa se ha demostrado que la LacNAc es transportada y fosforilada por el PTS de la lactosa e hidrolizada intracelularmente por la fosfo-b-galactosidasa LacG en galactosa-6P y GlcNAc. Análisis transcripcionales demostraron que el operon lac, además de por lactosa, está también inducido por LacNAc. Para intentar comprender mejor el metabolismo y potencial bioactivo de los OLH se necesitan cantidades adecuadas de éstos. Con el objeto de disponer de LNB y GNB en cantidad suficiente para ensayar su actividad biológica, se han sintetizado in vitro ambos disacáridos utilizando para ello la capacidad de transglicosidación de la glicosil hidrolasa GnbG aislada de L. casei. Las reacciones de transglicosidación se escalaron, los productos resultantes se purificaron, y se obtuvieron rendimientos de 10.7 ± 0.2 g/l de LNB y 10.8 ± 0.3 g/l de GNB. Ambos disacáridos fueron utilizados in vitro para determinar sus propiedades prebióticas potenciales con 33 cepas de Lactobacillus correspondientes a 13 especies diferentes. Se determinó que 21 de las cepas, correspondientes a las especies L. casei, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus gasseri y Lactobacillus johnsonii fueron capaces de metabolizar tanto GNB como LNB. Existen evidencias científicas recientes que les atribuyen a los OLH propiedades inmunomoduladoras, así que se han determinado éstas in vitro para LNB y GNB, y para dos fucosiloligosacáridos (Fuc-a-1,3-GlcNAc y Fuc-a-1,6-GlcNAc) sintetizados anteriormente en nuestro laboratorio. Se ha demostrado que los cuatro disacáridos son capaces de incrementar significativamente la producción de IFN-g en Células Mononucleares de Sangre Periférica. El Fuc-a-1,6-GlcNAc además fue capaz de reducir significativamente la producción de IL13. Estos resultados sugieren un efecto estimulante del sistema inmune y en el caso particular del Fuc-a-1,6-GlcNAc un efecto polarizador de la respuesta inmune Th1/Th2 / [CAT] La llet humana conté una gran quantitat d'oligosacàrids, lliures o conjugats amb lípids o proteïnes i la seua funció fisiològica és majoritàriament desconeguda. Aquests oligosacàrids són resistents a la digestió i una gran proporció arriba a l'intestí dels lactants, on poden ser substrat per a la microbiota. La lacto-N-biosa (LNB) i la galacto-N-biosa (GNB) són les estructures tipus-1 que conformen el nucli dels oligosacàrids de la llet humana (OLH) i de les glicoproteïnes de la mucina, respectivament. Els dos sucres són fermentats per espècies del gènere Bifidobacterium, però no existeixen dades sobre l'ús pel gènere Lactobacillus. Tampoc no hi ha informació per a aquest gènere sobre el metabolisme de la N-acetil-lactosamina (LacNAc), que cosntitueix la cadena de sucre tipus-2 als OLH i de la lacto-N-triosa, que forma part de l'estructura de als OLH tipus-1 i 2. La capacitat de L. casei, una bactèria làctica aïllada de múltiples nínxols ambientals, de prevaler al sistema gastrointestinal dependrà en part de la seua versatilitat metabòlica per a utilitzar els carbohidrats disponibles. A la present tesi doctoral s'ha demostrat que la soca L. casei BL23 es pot cultivar en presència de LNB, GBN, LacNAc i lacto-N-triosa com a fonts de carboni. Al metabolisme de la LNB, GNB i també de la N-acetilgalactosamina (GalNAc) està implicat l'operó gnbREFGBCDA. Anàlisi transcripcionals demostraren que l'operó gnb està regulat per inducció del substrat mitjançant el repressor transcripcional GnbR. A sobre de l'operó gnb, hi ha dos gens bnaG i manA, que codifiquen per al precursor d'una b-N-acetilglucosaminidasa i una manosa-6P isomerasa. S'ha demostrat que BnaG és un enzim extracel·lular unit a la paret cel·lular i que està implicat en el metabolisme de la lacto-N-triosa. Aquesta és hidrolitzada per BnaG en lactosa i N-acetilglucosamina (GlcNAc). L'enzim ManA està implicada en el metabolisme de la manosa present al disacàrid 3-N-acetilglucosaminil-manosa, el qual també representa una font de carboni per a L. casei BL23. Per concloure, en aquesta soca s'ha demostrat que la LacNAc és transportada i fosforilada pel PTS de la lactosa i hidrolitzada intracel·lularment per la fosfo-b-galactosidasa LacG en galactosa-6P i GlcNAc. Anàlisi transcripcionals demostraren que l'operó lac, a més de per lactosa està induït per LacNAc. Per intentar comprendre millor el metabolisme i potencial bioactiu dels OLH es necessiten quantitats adequades d'aquests. A fi de disposar de LNB i GNB en quantitats suficients per a assajar la seua activitat biològica, s'han sintetitzat in vitro ambdós disacàrids utilitzant per a tal la capacitat de trasglicosidació de la glicosil hidrolasa GnbG aïllada de L. casei. Les reaccions de transglicosidació s'escalaren, els productes resultants es purificaren i s'obtingueren rendiments de 10.7 ± 0.2 g/l de LNB y 10.8 ± 0.3 g/l de GNB. Ambdós disacàrids foren utilitzats in vitro per a determinar les seues propietats prebiòtiques potencials amb 33 soques de Lactobacillus corresponents a 13 espècies diferents. Es determinà que 21 de les soques, corresponents a les espècies L. casei, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus gasseri i Lactobacillus johnsonii foren capaces de metabolitzar tant GNB com LNB. Existeixen evidències científiques recents que atribueixen als OLH propietats immunomoduladores, així que s'han determinat aquestes in vitro per a LNB i GNB i per als dos fucosiloligosacàrids (Fuc-a-1,3-GlcNAc y Fuc-a-1,6-GlcNAc) sintetitzats anteriorment al nostre laboratori. S'ha demostrat que els quatre disacàrids són capaços d'incrementar significativament la producció de IFN-g en Cèllules Mononuclears de Sang Perifèrica. El Fuc-a-1,6-GlcNAc a més, fou capaç de reduir significativament la producció de IL13. Aquests resultats suggereixen un efecte estimulant del sistema immune i en el cas particular del Fuc-a-1,6-GlcNAc un efecte pol / Bidart Costoya, G. (2016). Metabolismo y síntesis de oligosacáridos de la leche humana mediante la utilización de enzimas glicosil hidrolasas de Lactobacillus casei [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/70898 / TESIS

Experiments were carried out to determine if pretreatment of cells with N-Methyl-N'-Nitro-N-Nitrosoguanidine (a potent alkylating agent known to induce DNA repair) would affect Herpes Simplex Virus Replication. The data demonstrated a 1.5 fold increase in virus yield, a 2 fold increase in HSV specific TK activity and no change in HSV specific DNA polymerase activity in MNNG treated cultures. The effects of MNNG treatment on virus replication and enzyme expression are discussed. / Thesis / Master of Science (MSc)