The primary objectives of this research were to characterize the involvement of lactate dehydrogenase (LDH) in color stability of physiologically different bovine muscles, and to investigate the influence of lactate enhancement on the myoglobin redox state of bovine muscles. In experiment 1, three different bovine muscles; Longissimus lumborum (LD), Semimembranosus (SM), Psoas major (PM) were (n=7 respectively) cut into steaks, and displayed for 7 days. Instrumental color, LDH-B, LDH isozyme expression, and NADH were measured. In experiment 2, strip steaks (n=8) were cut into half, and one side was injected with oxamate (LDH inhibitor), and the other was injected with water. Surface color, LDH, and NADH were measured after 10 days. In experiment 3, the three bovine muscles (n=10) were enhanced with solutions containing lactate and/or phosphate. Steaks were stored and displayed for 14 days. Instrumental color, LDH-B, total reducing activity (TRA), and NADH were measured. In experiment 4, fifteen beef strip loins were divided individually into four equal sections, and one of six treatments containing phosphate and/or calcium lactate with or without irradiation (2.4 kGy) randomly assigned to each loin section (n=10). Steaks were packaged in highoxygen modified atmosphere package, irradiated, stored in the dark at 1°C for 14 days. Instrumental color, TRA, lipid oxidation, and NADH were measured. LD remained the most red, whereas PM was most discolored. LD had a significantly higher level of LDH-1 responsible for LDH-B activity as compared to SM and PM. Consequently, LD had a higher LDH-B, and more NADH (p < 0.05). Inclusion of oxamate inhibited LDH-B, decreased NADH, and consequently discolored more. Potassium lactate enhancement led to more NADH through elevated LDH flux and subsequently increased (p < 0.05) color stability of LD and PM throughout display. Loins with calcium lactate/phosphate maintained the most stable red color during display. Calcium lactate/phosphate in loins increased NADH concentration, TRA, and were the least oxidized over display. These results confirm the involvement of LDH in meat color stability through replenishment of NADH. Lactate enhancement promotes meat color stability by providing superior antioxidant capacity and increased reducing activity of myoglobin by elevating NADH concentration.

No description available.

No description available.

Abstract Five species belonging to order Chelonia, two families and four genera, namely, Taiwanese soft-shelled turtle, Pelodiscus sinensis japonicus; American soft-shelled turtle, Apalone ferox; alligator snapping turtle, Macroclemys temminck; pitted shelled turtle, Carettochelys insculpta and side-necked turtle, Chelodina siebernrocki were investigated in order to fully understand the structural basis for the multiple lactate dehydrogenase (LDH) isozymes in turtles and soft-shelled turtles. Starch gel electrophoretic patterns of LDH isozymes from muscle, heart, liver, testis and eye were analyzed. Chelonia possess the two fundamental LDH loci-A¡]muscle¡^and B¡]heart¡^as the case of all other vertebrates. The major forms of LDH isozymes in the tissues of Chelonia are homotetrameric LDH-A4 and B4.While some of these Chelonia do not form two heterotetrameric A3B1 and/or A1B3 isozymes. This phenomenon is also observed among some lower vertebrates and fishes of other classes. I have determined the LDH-A and LDH-B cDNA sequences of protein-coding region from these five species. The 3D-structure of tetrameric LDH isozymes from Taiwanese soft-shelled turtle was predicted by homologous modeling and the substitutive residues in subunit contact sites were examined in oder to explain different multiple forms of tetrameric LDH isozymes present in various species. The LDH isozymes are housekeeping genes in most eukaryotic cells and therefore the LDH DNA or protein sequences can be an ideal marker for studying the molecular phylogenetics and evolution among different organisms. However, whether this marker can also be used to investigate the systematic relationship among the closely related species remains to be demonstrated. In this study, the newly determined LDH-A and LDH-B cDNA sequences and their deduced protein sequences from several different turtles and soft-shelled turtles, as well as the previously published LDH sequences, are analyzed by phylogenetic tree reconstruction methods of neighbor-joining, minimum evolution, maximum parsimony and maximum likelihood. These results confirm the traditional classification based on morphology of Chelonia as the two different families belonging to the same order Chelonia. Furthermore, these results clearly classify these Chelonia species into side-necked turtle, rough-shelled hide-necked turtle and smooth-shelled hide-neck turtle. Finally, these results also demonstrate that LDH can indeed be used as a molecular systematic marker for analyzing closely related species.

Ingeniero Civil Químico / El agua es de vital importancia para la subsistencia de seres vivos y de los ecosistemas, y su escasez hoy en día es tal que, en el peor de los escenarios, para el año 2050 afectará a un 72% de la población mundial. La contaminación industrial del agua es uno de los principales motivos, donde entre un 17 y un 20% de esa contaminación se debe a procesos de teñido de telas en la industria textil. Estos compuestos pueden ser eliminados, entre otros mecanismos, por fotocatálisis y adsorción. Se sintetizaron materiales con potencial uso en ambos mecanismos. Estos materiales corresponden a hidróxidos bilaminares (LDH) de Cobre con otros metales y fueron caracterizados mediante DRX, BET, DRS y SEM. Además, se buscó combinar estos LDH con grafeno oxidado (GO), buscando mejores resultados en cuanto a adsorción. LDH de Cobre/Aluminio con un alto grado de cristalinidad fueron sintetizados, logrando introducir Cobalto. Sin embargo, al reemplazar el Aluminio por Cromo, se obtuvo un material con baja cristalinidad. Estos LDH deben su capacidad de adsorción principalmente al intercambio iónico entre las moléculas de pigmento aniónico y las de CO32-, y al ser calcinados, la adsorción se rige por otro mecanismo, conocido como efecto de memoria . Ambos mecanismos fueron comprobados a través de DRX. Además, mediante SEM, se constató la permanencia de la estructura laminar al calcinar un LDH con alta cristalinidad. Los LDH con mejor desempeño fueron los calcinados, tanto de [CuCo]/Al como de Cu/Al, con capacidades máximas de adsorción de 583 y 780 [mg/g] respectivamente. Además, el [CuCo]/Al alcanza una efectividad de un 60% luego de ser utilizado 4 veces, regenerándose mediante tratamientos térmicos. Las pruebas de fotocatálisis no fueron fructíferas, ya que no mostraron una cinética distinta a la de adsorción, y, por otro lado, los LDH con GO, no mostraron la sinergia que se esperaba en la adsorción del naranjo de metilo.

There has been much interest in the use of therapeutics based on ribonucleic acid interference(RNAi) to inhibit synthesis of mutant proteins ever since Elbashir et al. (Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T., 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411, 494-498.) found that synthetic double stranded small interfering ribonucleic acids (siRNAs) can initiate this evolutionarily conserved process in mammalian cells. Since RNAi is able to target single genes and therefore mitigate the underlying molecular pathology of diseases, RNAi-based therapeutics will most likely benefit monogenic neurodegenerative diseases such as Huntington’s disease. It is however particularly difficult to deliver exogenous materials such as siRNAs into neurons in vivo as the blood-brain barrier (BBB) isolates the brain from the vascular system and prevents permeation of most materials. Neurons also do not take up exogenous materials readily. Therefore, effective delivery of siRNAs into the brain remains one of the biggest challenges impeding their use as a potential neurotherapeutic. Layered double hydroxide (LDH) nanoparticles are a class of anionic clay materials that have demonstrated great potential as a DNA (deoxyribonucleic acid) delivery system for a variety of mammalian cell lines due to their unique physiochemical properties. This thesis examined the feasibility of LDH as a siRNA delivery system for cultured neurons and demonstrated that the delivered siRNAs are able to effectively down-regulate synthesis of a target protein with minimal toxicity. Experiments were conducted using double stranded DNAs (dsDNAs) initially, and siRNAs were then used to verify these results. It was shown that nucleic acids(dsDNAs and siRNAs) could successfully intercalate into pristine LDHs to form nucleic acid-LDH complexes that had properties suitable for use as a delivery system in mammalian cells. These studies established that LDHs and nucleic acid-LDH complexes were biocompatible with neurons isolated from embryonic day 17.5 mouse cerebral cortex, suggesting that LDH can be used for nucleic acid delivery into cultured neurons. LDHs were also shown to successfully deliver nucleic acids into a non-neural mammalian cell line (NIH 3T3 cells). Finally, this thesis demonstrated for the first time that LDHs were able to deliver siRNAs into neurons, providing encouraging preliminary evidence that sequence specific gene silencing of the Mus Musculus Deleted in Colorectal Cancer (DCC) gene had occurred. However, down-regulation of the DCC protein did not occur consistently, suggesting that further optimisation is needed to improve the efficacy of siRNA-LDH complexes to inhibit expression of target protein in neurons. In future, LDHs should be further developed as an efficient siRNA delivery system for therapeutic gene silencing in the central nervous system using a neurodegenerative disease model such as the Huntington’s disease mouse model, which closely phenocopies the human disease. This model will allow the in vivo efficacy of these nanoparticles to be tested and subsequently improved in order to deliver siRNAs locally and systematically into the brain.

There has been much interest in the use of therapeutics based on ribonucleic acid interference(RNAi) to inhibit synthesis of mutant proteins ever since Elbashir et al. (Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T., 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411, 494-498.) found that synthetic double stranded small interfering ribonucleic acids (siRNAs) can initiate this evolutionarily conserved process in mammalian cells. Since RNAi is able to target single genes and therefore mitigate the underlying molecular pathology of diseases, RNAi-based therapeutics will most likely benefit monogenic neurodegenerative diseases such as Huntington’s disease. It is however particularly difficult to deliver exogenous materials such as siRNAs into neurons in vivo as the blood-brain barrier (BBB) isolates the brain from the vascular system and prevents permeation of most materials. Neurons also do not take up exogenous materials readily. Therefore, effective delivery of siRNAs into the brain remains one of the biggest challenges impeding their use as a potential neurotherapeutic. Layered double hydroxide (LDH) nanoparticles are a class of anionic clay materials that have demonstrated great potential as a DNA (deoxyribonucleic acid) delivery system for a variety of mammalian cell lines due to their unique physiochemical properties. This thesis examined the feasibility of LDH as a siRNA delivery system for cultured neurons and demonstrated that the delivered siRNAs are able to effectively down-regulate synthesis of a target protein with minimal toxicity. Experiments were conducted using double stranded DNAs (dsDNAs) initially, and siRNAs were then used to verify these results. It was shown that nucleic acids(dsDNAs and siRNAs) could successfully intercalate into pristine LDHs to form nucleic acid-LDH complexes that had properties suitable for use as a delivery system in mammalian cells. These studies established that LDHs and nucleic acid-LDH complexes were biocompatible with neurons isolated from embryonic day 17.5 mouse cerebral cortex, suggesting that LDH can be used for nucleic acid delivery into cultured neurons. LDHs were also shown to successfully deliver nucleic acids into a non-neural mammalian cell line (NIH 3T3 cells). Finally, this thesis demonstrated for the first time that LDHs were able to deliver siRNAs into neurons, providing encouraging preliminary evidence that sequence specific gene silencing of the Mus Musculus Deleted in Colorectal Cancer (DCC) gene had occurred. However, down-regulation of the DCC protein did not occur consistently, suggesting that further optimisation is needed to improve the efficacy of siRNA-LDH complexes to inhibit expression of target protein in neurons. In future, LDHs should be further developed as an efficient siRNA delivery system for therapeutic gene silencing in the central nervous system using a neurodegenerative disease model such as the Huntington’s disease mouse model, which closely phenocopies the human disease. This model will allow the in vivo efficacy of these nanoparticles to be tested and subsequently improved in order to deliver siRNAs locally and systematically into the brain.

No description available.

Le confinement des antibiotiques dans des matrices inorganiques, constituent une classe de matériaux particulièrement bien adaptée, pour la conservation de leurs activités. Cette thèse à pour objectifs: (1) d’établir une corrélation entre la localisation des antibiotiques dans les matrices HDL et leur activité antibactérienne, (2) de définir la matrice la plus adéquate pour protéger les antibiotiques de la dégradation thermique et/ou photonique. Nous nous sommes intéressés à étudier l’influence de la localisation de l’antibiotique dans le matériau sur son activité antibactérienne, et de vérifier si la phase HDL peuvent protéger les antibiotiques de la dégradation thermique et/ou photonique.Dans ces travaux, nous avons utilisé des hydroxydes doubles lamellaires (HDL) de type Zn2Al, Mg2Al, Ni2Al et Mn2Al comme des supports pour différentes types d’antibiotiques (cycline, polypeptide et enzymatique). Nous avons étudié l’immobilisation de deux antibiotiques de types cyclines tetracycline (TCH) et oxytetracycline (OXY), par deux méthodes (la coprécipitation et l’échange anionique). Nous avons étudié l’impact de l’irradiation du UV ou/et stockage à différentes températures (30, 60 et 120°C) pour évaluer leurs activités antibactériennes. Les résultats ont montré que les antibiotiques sont localisés à la surface des grains de la phase HDL. Les matériaux préparés par coprécipatation présentent un taux de relargage et une activité antibactérienne plus importante que celles préparé par échange anionique. Une baisse significative de l’activité antibactérienne après un stockage à haute température et l’exposition à la lumière UV est observée. Cela permet de conclure que la phase HDL accélère la dégradation des antibiotiques sous l’effet du température et la lumière UV. Nous avons étudié l’immobilisation de la nisine dans des différentes phases HDL, en tenant compte de l’effet du rapport molaire, l’effet de matrice, l’effet de l’anion et l’effet de morphologie. Les résultats ont montré que l’immobilisation de la nisine dans les différentes phases HDL n’affecte pas son activité antibactérienne à 4°C. En revanche, on remarque que l’activité de la nisine est moins sensible à la température quand elle est confinée.. La localisation de la nisine joue donc un rôle très important dans la meilleure protection de l’activité antibactérienne. Enfin nous avons effectué l’immobilisation du lysozyme dans les différentes phases HDL. Nos résultats ont montré que, le lysozyme est localisé à la surface des grains du matériau, on note un abattement plus important pour le lys libre que lYS adsorbé. Cela peut être du à une dénaturation du site enzymatique. / The confinement of antibiotics in inorganic matrices results in a class of materials particularly suitable for the conservation of their activities. This thesis aims to: (1) establish a correlation between the localization of antibiotics in HDL matrices and their antibacterial activity, (2) define the most appropriate matrix to prevent the antibiotics thermal and / or photon degradation. We were interested in studying the influence of the antibiotic localization in the material and the effect on the material antibacterial activity and whether the HDL phase can protect the antibiotics from thermal and / or photon degradation.In this work, we used double lamellar hydroxide (HDL) type Zn2Al, Mg2Al, Ni2Al and Mn2Al as supports for different types of antibiotics (cyclin, polypeptide and enzymatic). We investigated the immobilization of two cyclin types tetracycline (TCH) and oxytetracycline (OXY), by two methods (coprecipitation and anion exchange). We investigated the impact of UV irradiation and / or storage at different temperatures (30, 60 and 120°C) to evaluate their antibacterial activities. The results showed that the antibiotics are localized on the grain surface of the HDL phase. The materials prepared by coprecipitation have a higher release rate and antibacterial activity than those prepared by anion exchange. A significant decrease in the antibacterial activity after storage at high temperature and exposure to UV light was observed. This leads to the conclusion that the HDL phase accelerates the degradation of antibiotics under the effect of temperature and UV light. We investigated the immobilization of nisin in different HDL phases, taking into account the effect of the molar ratio, of the matrix, of the anion and the morphology. The results showed that the immobilization of nisin in the different HDL phases does not affect its antibacterial activity at 4°C. On the other hand, we noteiced that the activity of nisin is almost temperature sensitive under confinement. The localization of nisin plays therefore a very important rolefor the best protection of the antibacterial activity. Finally, we immobilized lysozyme in the various HDL phases. Our results showed that lysozyme is localized on the surface of the material grains, one notes a more important abatement for free lys than adsorbed LYS. This can be due to denaturation of the enzyme site.

L-Lactate dehydrogenases (LDH) are ¡§house keeping¡¨ enzymes. The LDH isozymes are known to be a very stable, slow-evolving and suitable to be a model to elucidate the phylogenetic relationships among various species. The cDNA sequences of LDH-A4 (muscle) and LDH-B4 (heart) from green iguana (Iguana iguana) were determined. The results of isozyme electrophoresis demonstrated that there are two isoforms of Iguana iguana LDH-B isozyme and called LDH-B and LDH-b. The protein structures of LDH-B and LDH-b monomer were constructed, and these two proteins have the same structure except the 5 different amino acids. The phylogenetic relationships between green iguana and other vertebrates, whose LDH cDNA sequences published previously, were analyzed by phylogenetic tree construction methods Mega2 program as well as Phylip program. These results on the relationships among lizards indicate that Iguana iguana is closer to Sceloporus woodi than to Sceloporus undulates. The divergent times between Iguana iguana and Sceloporus woodi, Iguana iguana and Sceloporus undulatus were estimated about 98 and 188 million years, respectively. The sequences of mitochondrial DNA(12S, 16S and ND1) among these three lizards were also analyzed, and the results were consistent with the traditional phylogenetics that species in the same genus were closer. The unexpected relationship that different genus of Iguana iguana and Sceloporus woodi is closer than that within same genus of Sceloporus woodi and Sceloporus undulates by analyzing among vertebrates LDH isozymes remained to be further confirmed.