Dissertations / Theses on the topic 'Osmolits'

En aquest estudi s'ha determinat que al augmentar el ritme d'extraccions de semen es produeixen canvis en el patró d'absorció i secreció del fluid epididimari, que provoquen alteracions en la maduració epididimaria dels espermatozoides i un desenvolupament anòmal de la motilitat espermàtica.
La concentració de glutamat i carnitina al fluid epididimari augmenten al llarg del conducte epididimari, alhora que la concentració de myo-inositol disminueix. El contingut de myo-inositol a l'interior dels espermatozoides disminueix, mentre que el contingut de glutamat augmenta a partir del caput distal i el contingut de carnitina no varia al llarg del conducte.
S'ha determinat la presència de la ruta del poliol a l'epidídim de porcí. Els resultats obtinguts indiquen que la glucosa difon de la sang cap al fluid epididimari, és convertida a sorbitol per l'aldosa reductasa, i aquest sorbitol s'acumula al fluid luminal i és convertit a fructosa per l'acció de la sorbitol deshidrogenasa.
A high semen collection frequency brought about an altered resorption and secretion pattern of the epididymal fluid, which results in defective sperm maturation and abnormal development of sperm motility.
In this study, it has been determined that in epididymal fluid the concentration of myo-inositol decreased in a proximo-distal direction, whereas intraluminal concentrations of L-carnitine and L-glutamate increased distally. The content of inositol in spermatozoa fell as they moved from the distal caput whereas sperm glutamate increased from the distal caput to more distal regions and carnitine content remained unchanged during epididymal transit.
In this study, evidence for an operative polyol pathway was demonstrated in the porcine epididymis. The results found are consistent with diffusion of circulating glucose into the lumen, its conversion via aldose reductase to sorbitol which accumulates in the lumen and the action of sorbitol dehidrogenase on sorbitol to produce fructose.

Living cells are composed of a variety of biological macromolecules such as nucleic acid, metabolites, proteins and cytoskeletal filaments as well as other particles. The fraction of the cellular interior volume that is taken by these biomolecules is about 30%, leading to a highly crowded environment. Biomolecules present in an extremely dense environment inside a cell have a completely different set of kinetic and thermodynamic behavior than in a test tube. Therefore comprehending the effect of crowding conditions on biological molecules is crucial to broad research fields such as biochemical, medical and pharmaceutical sciences. Experimentally, we are able to mimic such crowded environments; which are of more physiological relevance, by adding high concentrations of synthetic macromolecules into uncrowded buffers. Theoretically, very little attention has been paid to the effects of the dense cellular cytoplasm on biological reactions. The purpose of this work is to investigate analytically the effects of crowding agents on protein folding and stability. We present a new parameter as the measure of the polymer size, which will substitute the traditional measurements of the radius of gyration of the polymer and the end to end distance of a polymeric chain. Using this quantity we derive an expression for the free energy of the polymer which can easily be generalized to provide the free energy of a protein. This mechanism enables us to study the effect of crowding on folding and stability of a protein. The stabilization effects of the crowding particles depend on the concentration and the size of the crowders and also the type of the crowding particles that are present in the system. In our calculations the type of the crowders is controlled by the energetic parameter between the protein and surrounding macromolecules.

Au cours de ce travail nous avons montré que les osmolytes organiques tels que la glycine bétai͏̈ne (GB) inhibent l'activité de plusieurs enzymes dont celle de la glutamate déshydrogénase (GDH) et de la dihydrofolate réductase (DHFR). Cet effet est proportionnel à la concentration de l'osmolyte et présente les caractéristiques d'une inhibition incompétitive. Ces osmolytes organiques provoquent en effet une diminution de la vitesse de catalyse et une augmentation de l'affinité apparente pour les substrats. Au delà des paramètres optimaux des enzymes, ces dernières ne sont plus inhibées mais activées par la GB. Cette dualité d'effets est liée à la capacité de la GB à stabiliser les protéines et à réduire leur flexibilité. Nos résultats montrent que tous les solutés compatibles ont la capacité d'activer ou d'inhiber les enzymes, nous proposons un modèle qui, suivant les caractéristiques du soluté et de la protéine permet de comprendre cette dualité d'effet. Les agents déstabilisants tels que les sels, la température et l'urée ont un effet opposé à celui de la GB sur l'activité de la GDH. Leurs effets sont également intégrés au modèle proposé. L'inhibition de l'activité de la GDH contribuerait à réduire l'accumulation du glutamate lorsque la GB s'accumule dans la cellule. Ce phénomène permet ainsi d'expliquer la préférence des cellules pour la GB au détriment des solutés néosynthétisés lors de l'adaptation à un stress hyperosmotique. L'inhibition d'autres enzymes telles que la DHFR laisse présager un impact plus général de ces molécules sur le métabolisme cellulaire.

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Fundação de Amparo à Pesquisa do Estado de Minas Gerais
A papel da sacarose em CG foi investigado através da caracterização de plantas de Tabaco transgênicas superexpressando a isoforma 3 do gene sacarose sintase (NtSuSy3) sob controle do promotor KST1 bem como por experimentos de análise de fluxo metabólico utilizando fragmentos epidérmicos enriquecidos com CG (EF) durante abertura estomática induzida pela luz. As plantas NtSuSy3 mostraram aumentos na condutância estomática, fotossíntese e nas taxas de transpiração a nível foliar e de planta inteira. As alterações observadas no metabolismo de CG das plantas transgênicas são discutidas no texto. Observou-se uma redução nos níveis de sacarose em diferentes experimentos de abertura estomática induzida pela luz, enquanto que os níveis de sacarose no meio não foram alterados, sugerindo que a sacarose foi degradada no simplasto de CG. A análise via LC-qTOF-MS de experimentos com EF submetidos a NaH13C03 durante abertura estomática induzida pela luz mostraram um enriquecimento de 13 C em sacarose, malato, fumarate e glutamins. As possíveis funções exercidas por esses metabólitos são discutidas no texto. Em conjunto, os dados desse trabalho sugerem que a degradação da sacarose no simplasto de CG pode ser um mecanismo importante para a abertura estomática de Tabaco induzida pela luz.
A characterization of transgenic tobacco plants overexpressing potato sucrose synthase 3 gene (NtSuSy3) under control of KST1 promoter was performed in order to analyze the role of sucrose metabolism on GC osmoregulation. Also, we performed a metabolic flux analysis in guard cell enriched epidermal fragment (EF) of Nicotiana tabacum in order to investigate changes in GC metabolism during stomatal aperture light-induced. NtSuSy3 plants showed higher stomatal conductance, transpiration rate, whole plant transpiration, and net photosynthetic rate than wild type (WT). Several changes in GC metabolism were observed in transgenic plants are discussed in the text. In different stomatal aperture light-induced experiments, it was observed a decrease in sucrose content, while no changes were detected in the sugar content in the medium; suggesting that the sugars decreased observed is due to breakdown and not efflux of GC. Using a feeding strategy in EF submitted to NaH13C03 followed by LC-qTOF-MS analysis, a 13 C-enrichment in sucrose, malate, fumarate and glutamine were observed. The possible function of these metabolites for GC osmoregulation are discussed in the text. Taken together, the data showed here provide evidence for another role of sucrose for GC osmoregulation. Our data suggest that sucrose breakdown, not just sucrose accumulation, can be performed to induce stomatal opening in tobacco.
O autor não aprersentou título em portuguẽs. A data de aprovação foi aleatória devido não constar na tese. Tese enviada pela secretaria do curso por e-mail, em 28-03-17.

Einleitung: Mit der vorliegenden Studie wurden zum ersten Mal beim Menschen die Auswirkungen von Dehydration (Dehy) und anschließender Rehydratation (Rehy) auf cerebrale volumenregulatorische Metabolite (myo-Inosit (mI), N-Azetylaspartat+N-Azetylaspartylglutamat (tNAA), Kreatin (Cr), Glyzerophosphocholin+Phosphocholin (Cho) und Glutamat+Glutamin (Glx)) sowie auf Flüssigkeitsverschiebungen untersucht. Methoden: 14 Radsportler (26.6 (22.7/29.8) Jahre, Median und 25./75. Perzentile) wurden mittels 1H-Spektroskopie (1H-MRS) in der okzipitoparietalen grauen Substanz (GM) und parietalen weißen Substanz rechts (WMR) und links (WML) untersucht (GE Signa Horizon 3T94; PRESS: TE 30ms, TR 6000ms, VOI 8ml). Die Messungen erfolgten vor, direkt nach Dehy und nach Rehy (180min, Zufuhr von 150\% der verlorenen Körpermasse (KM)). Zusätzlich wurde durch T2-Relaxationsmessungen der Atrophieindex alpha (Verhältnis cerebrales Gewebewasser (HW) zu Liquor (CSF)) bestimmt. Resultate: Die KM der Probanden reduzierte sich durch Dehy um 3.7 (3.4/4.1)% und stieg durch Rehy wieder um 4.5 (3.7/5.3)% an (Wilcoxon: p
Introduction: In the present study the influence of Dehy on cerebral volume regulatory metabolites (myo-Inositol (mI), N-Acetyl-aspartata+N-Acetyl-aspartyl-glutamate (tNAA), Creatine (Cr), Glycerophosphocholine+Phosphocholine (Cho) and Glutamate+Glutamine (Glx)) and fluid shifts has been investigated for the first time in humans. Methods: 14 cyclists (26.6 (22.7/29.8) y, median and 25./75. percentile) have been examined with proton NMR spectroscopy in the occipito-parietal gray matter (GM) and the right (WMR) and left (WML) parietal with matter (GE Signa Horizon 3T94; PRESS: TE 30ms, TR 6000ms, VOI 8ml). Spectra were acquired before, immediately after Dehy and after rehydration (Rehy). Rehy took place during 180min and 150% of lost body weight (BW) was substituted. Additionally the atrophy index alpha (ratio between cerebral water and liquor) was assessed (T2 signal decay as a function of echo time). Results: BW of volunteers has been decreased 3.7 (3.4/4.1)% after Dehy and increased 4.5 (3.7/5.3)% after Rehy (Wilcoxon: p

It has previously been shown that high osmolarity is detrimental to cleavage-stage mouse embryo development in vitro, and that the presence of several organic osmolytes can provide protection against the detrimental effects of raised osmolarity. Whether the same is true for post-compaction stage embryos is unknown. In the present work, it was found that mouse post-compaction stage embryo development was inhibited by raised osmolarity. However, inhibition of embryo development from the 8-cell to the blastocyst stage occurred only at much higher osmolarities than that which inhibited development from the 1-cell stage. Glutamine, glycine, L-alanine and beta-alanine, which have been proven to function as organic osmolytes providing protection against increased osmolarity in cleavage-stage embryos (during the 1-cell through 4-cell stages), also protected post-compaction stage embryos from the detrimental effects of high osmolarity. Two other organic osmolytes, betaine and proline, which are effective in pre-compaction embryos, were not effective in providing protection against raised osmolarity in embryos developing in vitro from the 8-cell stage, nor were myo-inositol and taurine, which have been shown to be ineffective in cleavage-stage embryos. In addition, cleavage-stage embryos and post-compaction stage embryos were found to use different transport mechanisms to accumulate the four organic osmolytes that provided them with osmoprotection. When assessed in morulae, the amino acid transport system beta was found to be responsible for beta-alanine transport, while transport system B0+ mediated transport of glutamine, glycine and L-alanine. Glutamine, glycine, L-alanine and beta-alanine supported post-compaction stage embryo development, higher embryo cell number in blastocysts, and greater embryo volume at higher osmolarities. The four compounds identified do not share metabolic pathways or other such properties in common, and thus it is likely that post-compaction mouse embryos utilize them, in large part, as organic osmolytes.

By pulling and releasing the tension on protein homomers with the Atomic Force Miscroscope (AFM) at different pulling speeds, dwell times and dwell distances, the observed force-response of the protein can be fitted with suitable theoretical models. In this respect we developed mathematical procedures and open-source computer codes for driving such experiments and fitting Bell’s model to experimental protein unfolding forces and protein folding frequencies. We applied the above techniques to the study of proteins GB1 (the B1 IgG-binding domain of protein G from Streptococcus) and I27 (a module of human cardiac titin) in aqueous solutions of protecting osmolytes such as dimethyl sulfoxide (DMSO), glycerol and trimethylamine N-oxide (TMAO). In order to get a molecular understanding of the experimental results we developed an Ising-like model for proteins that incorporates the osmophobic nature of their backbone. The model benefits from analytical thermodynamics and kinetics amenable to Monte-Carlo simulation. The prevailing view used to be that small protecting osmolytes bridge the separating beta-strands of proteins with mechanical resistance, presumably shifting the transition state to significantly higher distances that correlate with the molecular size of the osmolyte molecules. Our experiments showed instead that protecting osmolytes slow down protein unfolding and speed-up protein folding at physiological pH without shifting the protein transition state on the mechanical reaction coordinate. Together with the theoretical results of the Ising-model, our results lend support to the osmophobic theory according to which osmolyte stabilisation is a result of the preferential exclusion of the osmolyte molecules from the protein backbone. The results obtained during this thesis work have markedly improved our understanding of the strategy selected by Nature to strengthen protein stability in hostile environments, shifting the focus from hypothetical protein-osmolyte interactions to the more general mechanism based on the osmophobicity of the protein backbone.

[ES] Durante siglos de cultivo en la Península Ibérica después de su introducción en el siglo XVI, las judías se adaptaron a nuevos entornos, evolucionando numerosas variedades locales. Se evaluaron cultivares españoles locales de Phaseolus lunatus (frijol lima) y su resistencia a la salinidad, en dónde se expusieron las plantas a varios tratamientos de sal, con el fin de evaluar el efecto de la salinidad en el crecimiento y el rendimiento del cultivo. Se observó que el estrés salino redujo el peso fresco de los órganos aéreos, lo que permitió clasificar los cuatro genotipos según su tolerancia a la salinidad. la prolina aumentó en todos los cultivares, más notablemente en el cv. VPH-79, con las concentraciones absolutas más altas registradas en los cultivares más tolerantes a la sal. Estos hallazgos indican que P. lunatus es moderadamente tolerante a la sal y que sus principales mecanismos para adaptarse al estrés salino son el mantenimiento de altas concentraciones de K+ y la acumulación de prolina en las hojas. Por otra parte, se analizaron en invernadero 24 genotipos locales de P. vulgaris de España durante dos temporadas consecutivas. De cada genotipo, se cultivaron cinco plantas y se caracterizaron (17 rasgos cuantitativos y 15 cualitativos) utilizando los descriptores del IBPGR. Los resultados obtenidos indican una alta variabilidad para la mayoría de los rasgos, especialmente los relacionados con el rendimiento y sus componentes. Además, se analizaron las respuestas a los tratamientos por déficit hídrico y estrés salino, en cuanto a inhibición del crecimiento y contenido de prolina foliar (Pro), en 47 genotipos de Phaseolus vulgaris de diferentes orígenes. Para la mayoría de las variables de crecimiento analizadas y Pro, los efectos del cultivo, el tratamiento y sus interacciones fueron altamente significativos (p<0.001); los rasgos morfológicos de las raíces, el diámetro del tallo y el número de hojas se debieron principalmente a una variación incontrolada, mientras que la variación del peso fresco y el contenido de agua de los tallos y las hojas fue inducida claramente por el estrés. Bajo las condiciones experimentales, los efectos promedio del estrés salino sobre el crecimiento de las plantas fueron relativamente más débiles que los del déficit hídrico. . Pro, por su parte, fue la única variable que mostró una correlación negativa con todos los parámetros de crecimiento, pero particularmente con los de tallos y hojas mencionados anteriormente, como lo indican los coeficientes de correlación de Pearson y los PCA. Se propone el uso de Pro como un marcador bioquímico adecuado para exámenes simples, rápidos y a gran escala de genotipos de judía, para excluir los más sensibles, aquellos que acumulan concentraciones más altas de Pro en respuesta a tratamientos de estrés hídrico o salino. Asimismo, se han analizado las respuestas a la salinidad en seis cultivares de judía común: cuatro variedades locales de España y dos líneas experimentales de Cuba. La prolina fue usada para clasificar la tolerancia de los cultivares, Las concentraciones de azúcares solubles totales variaron con los tratamientos y entre los genotipos, pero fue difícil evaluar su papel en la tolerancia al estrés de las plantas analizadas. Los cambios en el contenido de malondialdehído (MDA) no indicaron peroxidación de la membrana inducida por sal como resultado del estrés oxidativo secundario; en consecuencia, no se detectó acumulación de compuestos fenólicos totales y flavonoides, como mecanismo de defensa antioxidante. Estos resultados destacan la confiabilidad del uso de prolina como marcador bioquímico del estrés salino en judía y la importancia del mecanismo relacionado con el transporte de potasio a las hojas para conferir tolerancia al estrés a algunos cultivares de judía.
[CA] Durant segles de cultiu a la Península Ibèrica després de la seva introducció en el segle XVI, les mongetes es van adaptar a nous entorns, evolucionant nombroses varietats locals. Es van avaluar conreessis espanyols locals de garrofó (fesol llima) i la seva resistència a la salinitat, a on es van exposar les plantes a diversos tractaments de sal, per tal d'avaluar l'efecte de la salinitat en el creixement i el rendiment de l'cultiu. Es va observar que l'estrès salí va reduir el pes fresc dels òrgans aeris, el que va permetre classificar els quatre genotips segons la seva tolerància a la salinitat. la prolina augmentar en tots els conreessis, més notablement en el cv. VPH-79, amb les concentracions absolutes més altes registrades en els conreessis més tolerants a la sal. Aquestes troballes indiquen que P. lunatus és moderadament tolerant a la sal i que els seus principals mecanismes per adaptar-se a l'estrès salí són el manteniment d'altes concentracions de K + i l'acumulació de prolina en les fulles. D'altra banda, es van analitzar en hivernacle 24 genotips locals de P. vulgaris d'Espanya durant dues temporades consecutives. De cada genotip, es van conrear cinc plantes i es van caracteritzar (17 trets quantitatius i 15 qualitatius) utilitzant els descriptors de l'IBPGR. Els resultats obtinguts indiquen una alta variabilitat per a la majoria dels trets, especialment els relacionats amb el rendiment i els seus components. A més, es van analitzar les respostes als tractaments per dèficit hídric i estrès salí, pel que fa a inhibició de l'creixement i contingut de prolina foliar (Pro), en 47 genotips de Phaseolus vulgaris de diferents orígens. Per a la majoria de les variables de creixement analitzades i Pro, els efectes de l'cultiu, el tractament i les seves interaccions van ser altament significatius (p <0.001); els trets morfològics de les arrels, el diàmetre de la tija i el nombre de fulls es van deure principalment a una variació incontrolada, mentre que la variació de l'pes fresc i el contingut d'aigua de les tiges i les fulles va ser induïda clarament per l'estrès. Sota les condicions experimentals, els efectes mitjana de l'estrès salí sobre el creixement de les plantes van ser relativament més febles que els de el dèficit hídric. . Pro, per la seva banda, va ser l'única variable que va mostrar una correlació negativa amb tots els paràmetres de creixement, però particularment amb els de tiges i fulles esmentats anteriorment, com ho indiquen els coeficients de correlació de Pearson i els PCA. Es proposa l'ús de Pro com un marcador bioquímic adequat per a exàmens simples, ràpids i a gran escala de genotips de mongeta, per excloure els més sensibles, aquells que acumulen concentracions més altes de Pro en resposta a tractaments d'estrès hídric o salí. Així mateix, s'han analitzat les respostes a la salinitat en sis conreessis de mongeta comú: quatre varietats locals d'Espanya i dues línies experimentals de Cuba. La prolina va ser usada per a classificar la tolerància dels conreessis, Les concentracions de sucres solubles totals van variar amb els tractaments i entre els genotips, però va ser difícil avaluar el seu paper en la tolerància a l'estrès de les plantes analitzades. Els canvis en el contingut de malondialdehid (MDA) no van indicar peroxidació de la membrana induïda per sal com a resultat de l'estrès oxidatiu secundari; en conseqüència, no es va detectar acumulació de compostos fenòlics totals i flavonoides, com a mecanisme de defensa antioxidant. Aquests resultats destaquen la fiabilitat de l'ús de prolina com a marcador bioquímic de l'estrès salí en jueva i la importància de l'mecanisme relacionat amb el transport de potassi a les fulles per conferir tolerància a l'estrès a alguns conreessis de mongeta.
[EN] During centuries of cultivation in the Iberian Peninsula after their introduction in the 16th century, beans adapted to new environments, evolving numerous landraces.In this study was also evaluated the resistance to salinity of several local Spanish cultivars of Phaseolus lunatus L. (lima bean). Plants were subjected to various salt treatments and growth and biochemical parameters were determined. It was observed that salt stress reduced the fresh weight of aerial organs, which allowed us to classify the four genotypes according to their tolerance to salinity. In addition, proline increased in all cultivars, most notably in cv. VPH-79, with the highest absolute concentrations recorded in the most salt tolerant cultivars. These findings indicate that P. lunatus is moderately salt tolerant and that its main mechanisms for adapting to salt stress are the maintenance of high K+ concentrations and proline accumulation in leaves. In studies conducted in this research project, 24 landraces of P. vulgaris from Spain were analyzed in greenhouses during two consecutive seasons. From each genotype, five plants were grown and characterized for 17 quantitative and 15 qualitative traits using IBPGR descriptors. . The results obtained indicate high variability for most of the traits, especially those related to yield and its components. On the other hand, this study analyzed the responses to water deficit and salt stress treatments, in terms of growth inhibition and leaf proline (Pro) content, in 47 Phaseolus vulgaris genotypes of different origins. For most of the growth variables analyzed and Pro, the effects of cultivar, treatment and their interactions were highly significant (p <0.001); root morphological traits, stem diameter and number of leaves were mainly due to uncontrolled variation, whereas variation in fresh weight and water content of stems and leaves was clearly induced by stress. Under our experimental conditions, the average effects of salt stress on plant growth were relatively weaker than those of water deficit. . Pro, in turn, was the only variable that showed a negative correlation with all growth parameters, but particularly with those of stems and leaves mentioned above, as indicated by Pearson's correlation coefficients and PCAs. We propose the use of Pro as a biochemical marker suitable for simple, rapid, large-scale screening of bean genotypes to exclude the most sensitive, those that accumulate higher concentrations of Pro in response to water or salt stress treatments. In addition, responses to salinity were analyzed in six common bean cultivars: four local varieties from Spain and two experimental lines from Cuba. Proline was used to rank the relative tolerance of the cultivars. Concentrations of total soluble sugars varied with treatments and among genotypes, but it was difficult to assess their role in stress tolerance of the plants tested.. Changes in malondialdehyde (MDA) content did not indicate salt-induced membrane peroxidation as a result of secondary oxidative stress; consequently, accumulation of total phenolic compounds and flavonoids, as an antioxidant defense mechanism, was not detected. These results highlight the reliability of the use of proline as a biochemical marker of salt stress in common beans and the importance of the mechanism related to potassium transport to leaves in conferring stress tolerance to some common bean cultivars.
Arteaga Castillo, SM. (2021). Cultivos para el cambio climático: selección y caracterización de variedades de judía (Phaseolusvulgaris L.) y Phaseolus lunatus tolerantes a la sequía y salinidad [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/168450
TESIS

Despite extensive studies on Weddell seals (Leptonychotes weddellii) in McMurdo Sound since the 1960s, uncertainty still remains regarding female foraging habits during the lactation period. Based on their large body mass at the start of lactation and large relative mass loss at the end, the current hypothesis is that Weddell seals fast or feed to a neglible extent during lactation. However, this hypothesis has not been fully tested to date, as evidence for foraging is indirect and is based primarily on dive behaviour. The work presented in this thesis describes the development of a new dietary method, the biomarker method, and its application for studying the foraging behaviour of female Weddell seals during lactation. Biomarkers were used to (1) monitor the onset of feeding in individual animals, and (2) determine what prey females were feeding on using characteristic/taxon-specific biomarker patterns. Proton nuclear magnetic resonance spectroscopy (1H NMR) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed to detect and quantify dietary biomarkers in biological samples, mainly tissues, serum and plasma. Trimethylamine N-oxide, arsenobetaine, dimethylsulfoniopropionate, homarine and glycine betaine were first measured in thirty-three prey and potential prey species of Weddell seals collected from the Ross Sea and McMurdo Sound regions of Antarctica. These same compounds were then measured in the plasma of twelve female Weddell seals over the lactation period at the Hutton Cliffs seal colony, McMurdo Sound in 2006. Time-depth recorders monitored seal dive activity over the same period. The data obtained from both NMR and LC-MS/MS assays showed that biomarkers in Antarctic species varied both in content and concentration. The compound homarine, which occurs primarily in cephalopods, is suitable for distinguishing between major food groups of known prey of Weddell seals (i.e., fishes versus cephalopods). DMSP, a compound that occurs primarily in fish common in McMurdo Sound (e.g., Trematomus bernacchii and Pagothenia borchgrevinki) but not in significant amounts in Dissostichus mawsoni or Pleuragramma antarcticum, two main prey items for Weddell seals, may also be a suitable biomarker for distinguishing between major and minor prey types. The detection of plasma TMAO, AsB and homarine indicated that 75% of Weddell seals studied fed during lactation. The presence of these three compounds indicates the seals were preying upon a combination of fish and cephalopods. Two lactating females started foraging as early as 9 to 12 days postpartum and elevated biomarker levels were concurrent with increased dive activity. The onset of foraging and dive behaviour amongst individuals was highly variable; however, the results suggests that the number of females who feed during lactation may be more prevalent and initiated at an earlier stage than previously thought. This may have implications for future reproductive success given effects of climate change on sea ice abundance and resource availability. Overall, the work presented in this thesis provides new insights into the foraging behaviour of female Weddell seals during lactation and has added to the current knowledge of the biomarker distribution within the Antarctic ecosystem.

Thesis (Ph. D.)--Florida State University, 2009.
Advisor: W. Ross Ellington, Florida State University, College of Arts and Sciences, Dept. of Biological Science. Title and description from dissertation home page (viewed Sept. 30, 2009). Document formatted into pages; contains xi, 94 pages. Includes bibliographical references.

The preservation of water is critical for terrestrial organisms and the epidermis of skin is a major permeability barrier to water loss from within the body. Epidermal barrier function is provided not only by the stratum corneum but also by the presence of tight junctions (TJs). However, cellular mechanisms of water conservation such as osmolyte accumulation are also important, in particular by helping to maintain cell volume during times of cellular stress. Cellular mechanisms of water homeostasis are largely unexplored in skin and additionally, nothing is known regarding how cellular and extracellular mechanisms may interact. The aim of this study was to investigate the role of organic osmolytes in the control of TJ structure and function in the presence or absence of ultraviolet irradiation (UVR), a major source of water loss in skin. Data obtained from cell culture experiments showed that irradiation of cultured keratinocytes with UVB reduced tight junction (TJ) barrier function, which appeared to be due to dislocalisation of TJ proteins claudin-1 and claudin-4 and phosphorylation of occludin. The presence of organic osmolytes, betaine, taurine or myo-inositol, negated these effects without any change in the gene expression of TJ proteins which suggests that osmolytes affect TJs via a post-translational mechanism which also appears to be independent of effects on cell volume and production of reactive oxygen species (ROS) at least to some extent. Data acquired from human studies showed that human skin expresses the betaine, taurine and myo-inositol transporters and these have transporter specific expression patterns. Moreover, the expression of these transporters is regulated by UVB. Treatment of human skin with osmolytes in an ex vivo organ culture model resulted in increased expression of claudins-1 and -4 but not claudin isoforms -7 and -12. However, these molecules where found to use different mechanisms to induce these effects depending on the osmolyte. Betaine appeared to stabilise existing TJ proteins whereas taurine induced the synthesis of new TJ proteins. This preliminary study shows for the first time that organic osmolytes are not solely important for maintaining intracellular osmolarity and volume homeostasis but also modulate TJ integrity and mitigate the damaging effect of UVB, which could contribute to the barrier property of the epidermis.

Thesis advisor: Mary F. Roberts
Crystallization studies in presence of organic osmolytes were conducted to better understand the specific mechanism of compatible solute binding to the inositol monophosphatase of Archaeoglobus fulgidus. The synthesis of a-diglycerol phosphate, one of the natural osmolytes of A. fulgidus, was also completed for kinetic testing of its I-1-Pase thermoprotective properties and for crystallization trials
Thesis (MS) — Boston College, 2011
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry

The incidence of CF related diabetes is on the rise as patient life expectancy continues to improve. Sugars elevated in diabetics include glucose, fructose, and mannose. These sugars, in addition to mannitol (recently approved as an inhaled osmolyte) are the basis for this study, aimed at assessing the impact these clinically relevant sugars have on virulence in Burkholderia multivorans. B. multivorans is a member of the Burkholderia cepacia complex (Bcc), and is the most frequent cause of Bcc infection in CF patients. Using an exopolysaccharide-deficient knockout in macrophage and Galleria mellonella infection models, biofilm formation, and adhesion assays, this study has identified exopolysaccharide-dependent and -independent phenotypes. Sequencing of B. multivorans C1576, a CF outbreak isolate, identified three putative adhesins in clinical isolate C1576 but not present in the sequenced environmental strain ATCC17616. Mannitol promoted adhesion and enhanced expression of these adhesins. This study characterised these adhesins and assessed the distribution within other clinical and environmental isolates of B. multivorans and the Bcc. Additionally, transcriptomic profiling of B. multivorans assessed the sugar response and EPS regulation during growth on clinically relevant sugars. Where possible, links were made between phenotypic studies and transcriptome data. B. multivorans EPS derived from fructose and mannitol was subjected to composition analysis using mass spectrometry, and assessed for biological activity. Still relevant to CF related diabetes, the ability of some members of the Bcc to bind insulin was assessed. Results indicated that a minority of strains bound insulin. Furthermore, by using flow cytometry cell sorting and fluorescence microscopy, results also showed only a small number of cells within a given population that bound insulin. In all, this study has added to the knowledge base of B. multivorans but more work is needed to fully understand virulence strategies exploited by this CF pathogen.

Osmolytes affect protein stability through direct interactions with a protein, indirectly by perturbing the properties of the solvent water, and by a combination of these. In this thesis the conformational stability of the colicin E9 immunity protein (Im9) and Human serum albumin (HSA) were determined in the absence and presence of osmolytes (trehalose, sucrose, and glycerol) and their effective hydrodynamic radii measured in order to further explore the mechanism of stabilisation. Urea1/2, the midpoint of the unfolding transition, and GH2O, the free energy of unfolding, were measured in a urea-induced denaturation experiment and detected with fluorescence spectroscopy, and hydrodynamic radii (Rh) were measured with pulsed-field gradient NMR. The unfolding curves of Im9 and HSA are shifted to higher urea concentration so that Urea1/2 and GH2O increased, as the osmolyte concentration was increased indicating that Im9 and HSA are more stable in the presence of osmolytes. The Rh of Im9 and HSA increased in the presence of high concentration of trehalose and sucrose but glycerol produced a reduction. My data support the view that trehalose and sucrose act via a preferential hydration mechanism in which the water layer around the protein increases because osmolytes are excluded from the protein surface. In contrast, glycerol acts by interacting directly with the protein surface, and possibly by penetrating it, which causes the protein to become more compact as its void volume is reduced. This increase in compactness induces stabilisation. In addition, HSA formulations were studied for their stability over 6 months under various conditions using trehalose, sucrose and glycerol as stabilisers instead of acetyltryptophan and sodium octanoate, which are used commercially. However, measurements of HSA esterase-like activity and heme binding, and its aggregation state with polyacrylamide gel electrophoresis and DLS showed the osmolytes cannot stabilize the protein under high storage temperatures.

Myocilin, expressed in the trabecular meshwork of the eye, has been linked to inherited primary open-angle glaucoma (POAG). The biological function of myocilin is unknown, but mutant myocilin exhibits a gain-of-function mechanism, aggregating within the endoplasmic reticulum of human trabecular meshwork cells, causing cell stress and eventually apoptosis. After apoptosis occurs, the trabecular meshwork is compromised, leading to an increase in intraocular pressure, a symptom of glaucoma. In this thesis, I have expressed and purified the wild-type olfactomedin (OLF) domain and 24 reported disease-causing variants. I developed a facile thermal stability assay using differential scanning fluorimetry, which follows the unfolding of a protein through the fluorescence of a dye sensitive to hydrophobic regions of a protein. Also in this thesis I have determined melting temperatures for the wild-type and for each of the disease-causing mutants. I have tested the stability of the mutants in the presence of seven osmolytes, with sarcosine and trimethylamine-N-oxide restoring the melting temperature closest to wild-type. Additionally, I expressed and purified three reported single nucleotide polymorphisms (SNPs) (E352Q, E396D, K398R), which are considered benign variants. Variants were also compared by circular dichroism, revealing high b-sheet content and wild-type structure. When compared to previous studies, there is a positive correlation between the melting temperature, and previously reported qualitative assays, which measure the mutant myocilin solubility in detergent, secretion from mammalian cells, and aggregation propensity. Taken together, these data give insight into the relationship between glaucoma genotypes and phenotypes.

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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico
The screening for genes in metagenomic libraries from soil creates opportunities to explore the enormous genetic and metabolic diversity of microorganisms. Rivers are ecosystems with high biological diversity, but few were examined using the metagenomic approach. With this objective, a metagenomic library was constructed from DNA soil samples collected at three different points along the Jundia?-river (Rio Grande do Norte-Brazil). The points sampled are from open area, rough terrain and with the direct incidence of sunlight. This library was analyzed functionally and based in sequence. For functional analysis Luria-Bertani solid medium (LB) with NaCl concentration varied from 0.17M to 0.85M was used for functional analysis. Positives clones resistant to hypersaline medium were obtained. The recombinant DNAs were extracted and transformed into Escherichia coli strain DH10B and survival curves were obtained for quantification of abiotic stress resistance. The sequences of clones were obtained and submitted to the BLASTX tool. Some clones were found to hypothetical proteins of microorganisms from both Archaea and Bacteria division. One of the clones showed a complete ORF with high similarity to glucose-6-phosphate isomerase which participates in the synthesis of glycerol pathway and serves as a compatible solute to balance the osmotic pressure inside and outside of cells. Subsequently, in order to identify genes encoding osmolytes or enzymes related halotolerance, environmental DNA samples from the river soil, from the water column of the estuary and ocean were collected and pyrosequenced. Sequences of osmolytes and enzymes of different microorganisms were obtained from the UniProt and used as RefSeqs for homology identification (TBLASTN) in metagenomic databases. The sequences were submitted to HMMER for the functional domains identification. Some enzymes were identified: alpha-trehalose-phosphate synthase, L-ectoina synthase (EctC), transaminase L-2 ,4-diaminobutyric acid (EctB), L-2 ,4-diaminobutyric acetyltransferase (EctA), L-threonine 3 dehydrogenase (sorbitol pathway), glycerol-3-phosphate dehydrogenase, inositol 3-phosphate dehydrogenase, chaperones, L-proline, glycine betaine binding ABC transporter, myo-inositol-1-phosphate synthase protein of proline simportadora / PutP sodium-and trehalose-6-phosphate phosphatase These proteins are commonly related to saline environments, however the identification of them in river environment is justified by the high salt concentration in the soil during prolonged dry seasons this river. Regarding the richness of the microbiota the river substrate has an abundance of halobacteria similar to the sea and more than the estuary. These data confirm the existence of a specialized response against salt stress by microorganisms in the environment of the Jundia? river
A busca por genes baseada na constru??o e an?lise de bibliotecas metagen?micas a partir de solo gera oportunidades para explorar uma enorme diversidade gen?tica e metab?lica de microrganismos. Os rios s?o ecossistemas com alta diversidade biol?gica, mas ainda pouco explorados por meio de metagen?mica. Com o objetivo de explorar a diversidade microbiana, uma biblioteca metagen?mica foi constru?da a partir de DNA extra?do de substrato de rio em tr?s pontos ao longo do rio Jundia? (Rio Grande do Norte-Brasil). Os pontos de amostragem s?o derivados de ?rea aberta, terreno acidentado e com a incid?ncia direta da luz solar. Esta biblioteca foi analisada funcionalmente e tamb?m com base em sequ?ncias. Para a an?lise funcional foi utilizado meio de cultura s?lido LB com concentra??o de NaCl variando de 0,17M a 0,85M. Foram obtidos 15 clones positivos com caracter?sticas halotolerantes. Os DNAs recombinantes foram extra?dos e retransformados em cepa de Escherichia coli DH10B e curvas de sobreviv?ncia foram obtidas para confirma??o e quantifica??o da resist?ncia ao estresse abi?tico. As sequ?ncias dos clones foram obtidas e submetidas a ferramenta BLASTX e assim foi comprovado que alguns clones codificavam prote?nas hipot?ticas. Um dos clones apresentou uma ORF completa com elevada similaridade de glucose-6-fosfato-isomerase que participa na s?ntese do precursor de glicerol, sendo um soluto compat?vel para equilibrar a press?o osm?tica no interior e no exterior das c?lulas. Posteriormente, para identifica??o de genes que codificam osm?litos relacionados com halotoler?ncia e identifica??o da diversidade microbiol?gica, amostras de DNA ambiental do substrato do rio e da coluna d??gua do estu?rio e oceano foram coletadas e pirosequenciadas. As sequ?ncias de osm?litos de diferentes microrganismos foram obtidas a partir do UniProt e utilizadas como RefSeqs para a identifica??o por homologia (TBLASTN) nos bancos de dados metagen?micos. As sequ?ncias identificadas nos bancos de dados ambientais foram submetidas ao programa HMMER com o fim de identificar dom?nios funcionais. Foram identificadas as enzimas: alfa-trealose-fosfato sintase, L-ectoina sintase (ectC), transaminase do ?cido L-2,4-diaminobut?rico (EctB), ?cido L-2 ,4-diaminobut?rico acetiltransferase (EctA), L-treonina 3-desidrogenase (via de s?ntese do sorbitol), Glicerol-3-fosfato desidrogenase, inositol-3-fosfato desidrogenase, chaperonas, L-prolina glicina beta?na liga??o transportador ABC, mio-inositol-1-fosfato sintase, a prote?na simportadora de prolina/s?dio -PutP e trealose-6-fosfato fosfatase. Estas s?o enzimas que participam da s?ntese de osm?litos comumente relacionados a ambientes salinos, no entanto a identifica??o desses solutos em ambiente de rio ? justificada pela elevada concentra??o salina no solo durante prolongadas esta??es de seca neste rio. Quanto ? riqueza da microbiota foi identificado que o substrato do rio possui uma abund?ncia de halobact?rias semelhante a do mar e superior a do estu?rio. Esses dados confirmam a exist?ncia de uma resposta especializada contra o estresse salino por microrganismos no ambiente do rio Jundia?

>Magister Scientiae - MSc
Abiotic stress, mainly in the form of extreme temperatures, drought and salinity has caused major crop losses worldwide, putting a severe strain on agriculture. Salinity severely limits plant growth and productivity and affects all aspects of the plant’s development including the most crucial stage; germination. This study investigated the effect of salt (NaCl) stress on Sorghum bicolor seedlings and the role of exogenously applied calcium (Ca2+) to ameliorate the effects of salt stress during germination. Sorghum seeds were germinated in the presence and absence of various NaCl (100, 200 and 300 mM) and Ca2+ (5, 15 and 35 mM) concentrations. Several assays including physiological (germination and growth assays), biochemical (osmolytes and oxidative stress markers), anatomical (epidermal and xylem layers) and expression profiles of key genes [antioxidant (SbSOD, SbAPX2 and SbCAT3), Salt Overly Sensitive (SbSOS1, 2 and 3) pathway enzymes and the vacuolar Na+/H+ exchanger antiporter2 (SbNHX2)] were investigated. Salt stress delayed germination and negatively affected growth as observed by the reduced root and shoot length and decreased fresh and dry weight. There was an increase in proline content and oxidative stress markers (H2O2 and MDA) under salt stress. Oxidative stress resulted in damage to the epidermal and xylem layers as observed on Scanning Electron Microscopy (SEM) images. Quantitative real-time polymerase chain reaction revealed that salt stress induced the expression of SbAPX2, SbCAT3 and SbSOS1 genes, whereas SbSOD4A, SbSOS2, SbSOS3 and SbNHX2 genes were not affected by salt. Exogenous application of Ca2+ counteracted the harmful effects of salt stress by improving germination efficiency, promoting seedling growth, reducing oxidative damage and the Na+/K+ ratio, indicating the protective effect. Ca2+ also effectively protected the epidermis and xylem layers from the severe damage caused by salt stress. In the presence of Ca2+ the expression of SbAPX2 and SbCAT3 was reduced except for the SbNHX2 gene, which increased by 65-fold compared to the control. The results obtained suggests that sorghum is able to respond to salt stress by inducing osmolytes, the antioxidant defence system as well as the SOS pathway. Furthermore, 5 mM Ca2+ was determined as the optimum Ca2+ concentration required to enhance sorghum’s tolerance to salt stress.

[EN] Abstract Introduction Salinity and drought are the most important environmental stress conditions reducing crop yields worldwide and limiting the distribution of wild plants in nature. Soil salinity, especially secondary salinity caused by anthropogenic practices, such as prolonged irrigation, lead to substantial agricultural yield losses, especially in arid and semiarid regions. Drought, caused by reduced water content in the soil, occurs due to disorders in nature's water cycle, chiefly when evapotranspiration exceeds precipitation in a certain area, to the point where soil water reserves can no longer support plant growth. Drought and salt stress trigger the activation of a series of basic stress mechanisms that includes among others, the control of ion transport, exclusion and compartmentalization, as well as the accumulation of compatible solutes ('osmolytes'), and the activation of antioxidant systems. These mechanisms are conserved in all plants, stress tolerant and sensitive alike, and don't necessarily confer tolerance. To decipher those mechanisms and have a better understanding on the contribution of different stress responses to the stress tolerance of a given species, we have carried out comparative studies on the responses to drought and salinity in a number of genetically related taxa with different tolerance potentials. Methodology The experimental approach was mostly based on i) establishing the relative tolerance to water and salt stress in the studied species from their distribution in nature (in the case of wild species) and through the relative inhibition of growth in the presence of stress, and ii) correlating changes in the levels of biochemical 'stress markers' associated to specific response pathways (ion transport, osmolyte accumulation¿) upon stress treatments, with the already established relative tolerance to stress. This strategy proved to be appropriate to distinguish mere general responses to stress from those mechanisms relevant for stress tolerance of the investigated species and cultivars. The work also sheds light on other aspects affected by salt stress, specifically regarding germination and reproductive success or anatomical changes in salt-stressed plants. The expression patterns of the gene NHX1, encoding a vacuolar Na+/H+ antiporter were also studied in the Plantago taxa, as a first step in the full characterisation of this ion transporter, that appears to play an important role in the mechanisms of salt tolerance in this genus. Conclusion The results obtained in this work contribute to a better understanding of general stress tolerance mechanisms in plants, and provides clear insights into the mechanisms conferring tolerance, specifically, to drought and salt stress in some wild species and crops. This work also shed more light on the highly efficient responses to stress in halophytes, plants that could be viewed as nature's answer to the aforementioned adverse environmental conditions via evolution and adaptation. Halophytes can therefore be considered as a suitable source - underutilized at present, in our opinion - of knowledge, genetic resources and biotechnological tools for the needed improvement of stress tolerance in crops.
[ES] Resumen Introducción La salinidad y la sequía son las condiciones de estrés ambiental más importantes, que reducen los rendimientos de los cultivos en todo el mundo y que limitan la distribución de las plantas silvestres en la naturaleza. La salinidad del suelo, especialmente la salinización secundaria causada por prácticas antropogénicas, como la irrigación prolongada, conducen a pérdidas importantes de rendimiento agrícola, especialmente en las regiones áridas y semiáridas. La sequía, provocada por la reducción de contenido de agua en el suelo, se produce debido a alteraciones en el ciclo del agua en la naturaleza, principalmente cuando la evapotranspiración excede la precipitación en un área determinada, hasta el punto que las reservas de agua del suelo ya no pueden soportar el crecimiento de la planta. La sequía y el estrés salino desencadenan la activación de una serie de mecanismos básicos de respuesta, que incluyen entre otros el control del transporte, la exclusión y la compartimentación de iones, así como la acumulación de solutos compatibles ('osmolitos'), y la activación de sistemas antioxidantes. Estos mecanismos están conservados en todas las plantas, tolerantes y sensibles a estrés por igual, y no confieren necesariamente tolerancia. Para descifrar estos mecanismos y conseguir una mejor comprensión de la contribución de diferentes respuestas a estrés a la tolerancia al estrés en una especie dada, hemos llevado a cabo estudios comparativos sobre las respuestas a la sequía y la salinidad, en un número de taxones relacionados genéticamente con diferentes potenciales de tolerancia. Metodología El enfoque experimental se basó principalmente en i) establecer la tolerancia relativa al estrés hídrico y al estrés salino en las especies estudiadas, a partir de su distribución en la naturaleza (en el caso de especies silvestres) y atendiendo a la inhibición relativa de su crecimiento en presencia de estrés, y ii) correlacionar cambios en los niveles de 'marcadores bioquímicos de estrés' asociados a vías específicas de respuesta (transporte de iones, acumulación de osmolitos ...) inducidos por los tratamientos de estrés, con la tolerancia relativa a estrés de las plantas, previamente establecido. Esta estrategia ha resultado ser apropiada para distinguir meras respuestas generales a estrés de los mecanismos relevantes para la tolerancia a estrés de las especies y cultivares investigados. El trabajo también arroja luz sobre otros aspectos afectados por el estrés salino, específicamente en relación con la germinación y el éxito reproductivo, o cambios anatómicos en las plantas tratadas con sal. También se estudiaron los patrones de expresión del gen NHX1, que codifica un antiportador vacuolar Na+/H+, en las especies de Plantago, como un primer paso en la caracterización completa de este transportador de iones, que parece desempeñar un papel importante en los mecanismos de tolerancia a sal en este género. Conclusión Los resultados obtenidos en este trabajo contribuyen a una mejor comprensión de los mecanismos generales de tolerancia al estrés en plantas, y proporcionan ideas claras sobre los mecanismos que confieren tolerancia, en concreto, a la sequía y al estrés salino, en algunas especies silvestres y cultivadas. Este trabajo también arroja más luz sobre las respuestas a estrés altamente eficientes en halófitas, plantas que podrían ser vistas como la respuesta de la naturaleza a las condiciones ambientales adversas antes mencionadas, a través de la evolución y la adaptación. Por lo tanto, las halófitas pueden ser consideradas como una fuente adecuada - infrautilizada en la actualidad, en nuestra opinión - de conocimiento, recursos genéticos y herramientas biotecnológicas para la necesaria mejora de la tolerancia al estrés en plantas cultivadas.
[CA] Resum Introducció La salinitat i la sequera són les condicions d'estrès ambiental més importants, que redueixen els rendiments dels cultius a tot el món i que limiten la distribució de les plantes silvestres en la naturalesa. La salinitat del sòl, especialment la salinització secundària causada per pràctiques antropogèniques, com la irrigació perllongada, condueixen a pèrdues importants de rendiment agrícola, especialment en les regions àrides i semiàrides. La sequera, provocada per la reducció de contingut d'aigua en el sòl, es produeix a causa d'alteracions en el cicle de l'aigua en la naturalesa, principalment quan la evapotranspiració excedeix la precipitació en un àrea determinada, fins al punt que les reserves d'aigua del sòl ja no poden suportar el creixement de la planta. La sequera i l'estrès salí desencadenen l'activació d'una sèrie de mecanismes bàsics de resposta, que inclouen entre uns altres el control del transport, l'exclusió i la compartimentació d'ions, així com l'acumulació de soluts compatibles ('osmolits'), i l'activació de sistemes antioxidants. Aquests mecanismes estan conservats en totes les plantes, tolerants i sensibles a estrès per igual, i no confereixen necessàriament tolerància. Per a desxifrar aquests mecanismes i aconseguir una millor comprensió de la contribució de diferents respostes a estrès a la tolerància a l'estrès en una espècie donada, hem dut a terme estudis comparatius sobre les respostes a la sequera i la salinitat, en un nombre de taxons relacionats genèticament amb diferents potencials de tolerància. Metodologia L'enfocament experimental es va basar principalment en i) establir la tolerància relativa a l'estrès hídric i a l'estrès salí en les espècies estudiades, a partir de la seua distribució en la naturalesa (en el cas d'espècies silvestres) i atenent a la inhibició relativa de el seu creixement en presència d'estrès, i ii) correlacionar canvis en els nivells de 'marcadors bioquímics d'estrès' associats a vies específiques de resposta (transport d'ions, acumulació d'osmolits ...) induïts pels tractaments d'estrès, amb la tolerància relativa a estrès de les plantes, prèviament establert. Aquesta estratègia ha resultat ser apropiada per a distingir meres respostes generals a estrès dels mecanismes rellevants per a la tolerància a estrès de les espècies i conreus investigats. El treball també llança llum sobre altres aspectes afectats per l'estrès salí, específicament en relació amb la germinació i l'èxit reproductiu, o canvis anatòmics en les plantes tractades amb sal. També es van estudiar els patrons d'expressió del gen NHX1, que codifica un anti-portador vacuolar Na+/H+, en les espècies de Plantago, com un primer pas en la caracterització completa d'aquest transportador d'ions, que sembla exercir un paper important en els mecanismes de tolerància a sal en aquest gènere. Conclusió Els resultats obtinguts en aquest treball contribueixen a una millor comprensió dels mecanismes generals de tolerància a l'estrès en plantes, i proporcionen idees clares sobre els mecanismes que confereixen tolerància, en concret, a la sequera i a l'estrès salí, en algunes espècies silvestres i conreades. Aquest treball també llança més llum sobre les respostes a estrès altament eficients en halòfites, plantes que podrien ser vistes com la resposta de la naturalesa a les condicions ambientals adverses abans esmentades, a través de l'evolució i l'adaptació. Per tant, les halòfites poden ser considerades com una font adequada - infrautilitzada en l'actualitat, en la nostra opinió - de coneixement, recursos genètics i eines biotecnològiques per a la necessària millora de la tolerància a l'estrès en plantes conreades.
Al Hassan, M. (2016). Comparative analyses of plant responses to drought and salt stress in related taxa: A useful approach to study stress tolerance mechanisms [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61985
TESIS

A L- asparaginase é uma enzima aplicada no tratamento de Leucemia Linfoide Aguda, que atua na hidrólise da L- asparagina, privando a célula tumoral de um aminoácido essencial para o seu crescimento. A L- asparaginase, como outros biofármacos, deve ser estável, manter sua atividade específica e formar poucos agregados. A fim de manter a integridade do biofármaco, são utilizados adjuvantes nas formulações farmacêuticas, e dentre os mais importantes estão os osmólitos. Essas moléculas protegem a estrutura nativa da proteína, sendo capazes de interferir na formação de agregados e garantir a estabilidade proteica. O presente trabalho teve o objetivo de estudar o efeito dos osmólitos sacarose, sorbitol, arginina e glicina na atividade específica, estabilidade, cinética e caracterização de agregados na solução de L- asparaginase II de Erwinia chrysanthemi. Os resultados mostraram que a maioria dos osmólitos testados aumentou a atividade específica e a estabilidade da enzima, o que pode estar relacionado com o aumento da velocidade máxima e do kcat observados no ensaio cinético realizado com sacarose e sorbitol. Um perfil diferente de agregados foi encontrado para cada tipo de osmólito. A presença de sacarose ou sorbitol resultou na menor quantidade de agregados na faixa de, respectivamente, 100 a 200 e 200 a 300 nm em relação a enzima sem osmólito. Por outro lado, aumento no número total de agregados e presença de moléculas de alto peso molecular (300 a 500 nm) foram observados nas soluções enzimáticas contendo, respectivamente, glicina e arginina. Dessa forma, os resultados obtidos neste trabalho poderão auxiliar na produção e escolha da formulação de biofármacos, e, consequentemente, melhorar o tratamento medicamentoso de pacientes.
L L-Asparaginase is an enzyme applied in the treatment of Acute Lymphoblastic Leukemia, which acts on the hydrolysis of L- asparagine, depriving the tumor cell of an essential amino acid for its growth. L-asparaginase, as other biopharmaceuticals, must be stable, maintain its specific activity and form few aggregates. In order to maintain the integrity of the biopharmaceutical, adjuvants are used in the pharmaceutical formulations, and among the most importants adjuvants are the osmolytes. These molecules protect the native structure of the protein, being able of interfering in the formation of aggregates and guarantee protein stability. The present work had the objective of studying the effect of the osmolytes sucrose, sorbitol, arginine and glycine in the specific activity, stability, kinetic and aggregates characterization, in L- asparaginase II solution of Erwinia chrysanthemi. The results showed that the majority of the tested osmolytes increased the specific activity of the enzyme and its stability, which may be related to the augment of maximum velocity and kcat observed in the kinetic assay performed with sucrose and sorbitol. A different profile of aggregates was found for each type of osmolyte. The presence of sucrose or sorbitol resulted in the least amount of aggregates in the range of, respectively, 100-200 and 200-300nm in relation to the enzyme without osmolyte. On the other hand, increase in the total number of aggregates and the presence of high molecular weight molecules (300 to 500 nm) were observed in the enzymatic solutions containing, respectively, glycine and arginine. Thus, the results obtained in this work may help in the production and choice of the formulation of biopharmaceuticals and, consequently, improve the drug treatment of patients.

Pyrosequencing is a new DNA sequencing method relying on thesequencing-by-synthesis principle and bioluminometric detectionof nucleotide incorporation events. The objective of thisthesis was improvement of the Pyrosequencing method byincreasing the thermal stability of firefly luciferase, and byintroducing an alternative DNA polymerase and a new nucleotideanalog. Furthermore, the development of a new bioluminescentassay is described for the detection of inorganicpyrophosphatase activity.

The wild-type North American firefly(Photinus pyralis)luciferase is a heat-sensitiveenzyme, the catalytic activity of which is rapidly lost attemperatures over 30°C. Two strategies for increasing thethermostability of the enzyme are presented and discussed. Inthe first strategy, the solution thermodynamics of the systemis affected by osmolytes in such a way that heat-mediatedinactivation of the enzyme is prevented. In the secondstrategy, the enzyme is thermostabilized by mutagenesis. Bothstabilizing strategies can be utilized to allow bioluminometricassays to be performed at higher temperatures. For instance,both DNA polymerase and ATP sulfurylase activity could beanalyzed at 37°C.

The osmolyte strategy was successfully employed forincreasing the reaction temperature for the Pyrosequencingmethod. By increasing the reaction temperature to 37°Cunspecific signals from primer-dimers and 3’-end loopswere reduced. Furthermore, sequencing of a challenging templateat 37°C, which previously yielded poor, non-interpretablesequence signals at lower temperatures was now possible.

Introduction of a new adenosine nucleotide analog,7-deaza-2’-deoxyadenosine-5’-triphosphate (c⁷dATP) reduced the inhibitory effect on apyraseobserved with the currently used analog,2’-deoxyadenosine-5’-O-(1-thiotriphosphate)(dATPαS).

Sequencing of homopolymeric T-regions has previously beendifficult with the exonuclease-deficient form of the DNApolymerase I large (Klenow) fragment. By using the DNApolymerase from bacteriophage T7, known as Sequenase, templateswith homopolymeric T-regions were successfully sequenced.Furthermore, it was found that the strand displacement activityfor both polymerases was strongly assisted if the displacedstrand had a 5’-overhang. In contrast, the stranddisplacement activity for both polymerases was inhibitedwithout an overhang, resulting in reduced sequencingperformance in double stranded regions.

A firefly bioluminescent assay for the real-time detectionof inorganic pyrophosphatase in the hydrolytic direction wasalso developed. The assay is versatile and has a linearresponse in the range between 8 and 500 mU.

Key words:bioluminescence, osmolytes, glycine betaine,thermostability, firefly luciferase, inorganic pyrophosphatase,inorganic pyrophosphate, Pyrosequencing technology, secondaryDNA-structures, Sequenase, Klenow-polymerase, reaction rates,temperature, c⁷dATP, dATPαS.

Les osmolytes sont des solutés accumulés dans les cellules de nombreux organismes lors d'une contrainte hyperosmotique pour maintenir le volume cellulaire. Ils peuvent néanmoins induire une inhibition des enzymes dont les bases moléculaires sont au centre de nombreuses études. Nous avons étudié la DHFR, en présence de divers solutés. Les osmolytes ne modifient pas la structure globale de la DHFR et inhibent son activité tout en stabilisant sa structure. Cette inhibition est liée à la diminution du k[indice :]off du produit en présence d'osmolytes. L'étude de la dynamique interne de la DHFR apporte des réponses sur l'origine de l'inhibition de la DHFR en présence d'osmolytes. Une seconde partie présente nos travaux sur la relation structure-activité de peptides antimicrobiens. Ce projet s'inscrit dans la course au développement de nouvelles molécules actives pour substituer les antibiotiques conventionnels. Nous avons déterminé les structures de peptides en environnement membranaire modèle
The osmolytes are small molecules accumulated by cells of a wide variety of organisms in response to hyperosmotic stress to maintain the cellular volume. Nervetheless, enzyme activity is inhibited by these cosolutes and the molecular basis of their effects on the proteins properties is of great interest. We studied the DHFR in presence of the osmolytes. We demonstrate that its overall structure is maintained. While the osmolytes stabilize the DHFR structure, they inhibit its activity at the same time. The k[index :]off, of substrate analogues decreases with increasing the osmolyte concentrations and reflects the variation of DHFR catalytic rate. The study of the DHFR dynamic on several timescales gives answer of the origins of the DHFR inhibition in presence of osmolytes. The second chapter concerns the study of the structure-activity relationship of antimicrobial peptides. The main objective of this project is to develop new drugs. We solved NMR solution structure of in various model membranes

博士
國立中興大學
生命科學系所
100
The osmotic adaptation of halophilic methanogen, Methanohalophilus portucalensis FDF1T could uptake potassium or de novo synthesized glycine betaine, β-glutamine and Nε-acetyl-β-lysine as compatible solutes to encounter the increasing external osmotic and salt stress. Most importantly, it also posses an energy-required, high affinity and highly specific glycine betaine transport system. An ABC type transporter for glycine betaine, Bta (betaine transporter in Archaea) system, in M. portucalensis FDF1T has revealed and the transcription of bta expression was activated immediately in presence of external glycine betaine upon the salt stress or heat stress. In this study, the bta gene cluster was confirmed as btaA-btaB-btaC1-btaC1’---btaC2 by metagenomic pyrosequencing data of M. portucalensis FDF1T, which is the first ABC transporter containing three substrate binding proteins (SBP) genes from methanogic archaea. RT-PCR analysis revealed the possible post-transcriptional modification through two base splicing in btaA while cells under salt upshock and the betaine is available in the environment. The sequence analysis and protein homology modeling of BtaA revealed two CBS (cystathionine β-synthase) domains were located at C-terminal extended region, and surface-exposed four cationic residues in the CBS domains are critical for ion sensing. It suggested BtaA play the major role of osmotic regulation in Bta system. All three substrate binding proteins, BtaC1, BtaC1’ and BtaC2, contained a tryptophan prism for betaine binding as other known substrate betaine-binding proteins. The transcription of btaC1 and btaC2 were both activated upon the hyper-salt or cold stress, whereas the expression of btaC1 are 3-10 fold higher than btaC2. BtaC1SPD and BtaC1’SPD are a high affinity betaine specific binding protein, and could also bind dimethylglycine or sarcosine, respectively, and carnitine. The glycine betaine binding affinity of BtaC2SPD is low, but it could bind multiple substrates, such as dimethylglycine, sarcosine, choline and carnitine. The functional complementation analysis indicated BtaAB core transporter could recruited BtaC1 and BtaC2 to translocate osmolyes into cell. This investigation revealed the occurrence of multiple substrate-binding proteins for betaine transport system Bta to response the salt and temperature stresses and the broad substrate spectrum and substrate betaine affinity range suggested the delicate network between osmolyte betaine transport and biosynthesis in osmotic regulatory system.

碩士
國立中興大學
生命科學系
93
Methanohalophilus portucalensis strain FDF1 is one of the few organisms that can de novo synthesize osmolyte glycine betaine from glycine through threefold methylation. And S-adenosylmethionine (SAM) was verified as a methyl group donor. Glycine betaine is the common osmolyte and has the highest osmoprotection efficiency. The strategy for cloning the gene of the glycine betaine synthesizing enzyme was performed by the polymerase chain reaction technology (PCR) and the reverse transcription polymerase chain reaction technology (RT-PCR). Primers were designed from the fifteen amino acid sequences of the N-terminal glycine betaine synthesizing enzyme glycine sarcosine dimethylglycine N-methyltransferase (GSDMT) of M. portucalensis FDF1 and SAM binding motifs of glycine betaine de novo synthesizing enzymes from halophilic bacteria. The fragment RT-2 and RT-3, which was from RT-PCR with the degenerate primer from N-terminal amino acid sequences of GSDMT of M. portucalensis FDF1 and the oligo dT primer, was present only under the condition with no external glycine betaine addition. RT-2 and RT-3 contained fourteen N-terminal amino acid sequences of glycine betaine de novo synthesizing enzyme of M. portucalensis FDF1 and the N-terminal amino acid sequences of KINEA of chymotrypsin digested GNMT. The LC-MS/MS results showed that GSDMT contained the amino acid fragment similar to Methanosarcina barkeri dimethylamine methyltransferase MtbB1. And the amino acid fragment that similar to MtbB1 is also found in RT-2 and RT-3. Therefore the RT-2 and RT-3 amplified by RT-PCR are glycine betaine de novo synthesizing partial gene of M. portucalensis FDF1.

博士
國立中興大學
生命科學系
93
Strictly anaerobic methanogenic archaea are ubiquitous. Dr. Lai’s lab has isolated over twenty methanogens from Taiwan. Among them, strain N2F9704T was isolated from the seawater fish pond and strain P2F9704aT isolated from the estuarine near Wong-gong, Taiwan. Both strains are mesophilic, irregular cocci. Cells could metabolize formate and H2/CO2 as carbon source to obtain the energy through methanogenesis. Trace amounts of tungstate not only promoted growth of strain N2F9704T but also extended the range of growth conditions. However the trace element of tungstate only lightly stimulated the growth of strain P2F9704aT. Analyses of 16S rDNA sequences revealed that strain N2F9704T and strain P2F9704aT are members of genera Methanofollis and Methanocalculus respectively, and are named as Methanofollis aquaemaris N2F9704T and Methanocalculus taiwanensis P2F9704aT. Additionally, strain N2F9704T was infected with a novel coccus-shaped, enveloped virus MMV with a diameter of 200 nm. Both strains were the first published new methanogenic species from Taiwan. In addition to characterization of methanogens from Taiwan, the investigation of the osmoregulation of the betaine synthesizing halophilic methanogen, Methanohalophilus portucalensis FDF1, and the comparisons of osmoregulatory mechanisms between bacteria and archaea are our subject. Firstly, searching the bacterial osmoprotectants transporters through 25 archaeal genomes showed it may be the effect of the lateral gene transfer that archaea contain several homologues genes of bacterial osmoregulatory transporters, including proU, opuD and proP. Based on these informations, an ABC type transporter for betaine, the Bta (betaine transporter in Archaea) system, was identified in M. portucalensis FDF1. The gene structure of the bta gene was similar to proU of Escherichia coli. Transcriptional inducer tests revealed that betaine, choline and its de novo synthesized intermediates, glycine, sarcosine and dimethylglycine, could immediately increase the transcriptional level of the bta gene under the hyperosmotic stress. But proline and carnitine, the osmolytes occurred in bacteria, couldn’t increase the bta expression. Upon the heat shock, the transcription of the bta gene was also activated immediately in the presence of external glycine betaine. Moreover, the upstream sequences of the bta gene have conserved sequences similar to other archaeal heat shock responsed genes, like dnaK and grpE. It suggests that in addition to play a role in osmotic stress, bta also responds to the heat stress. This ABC type glycine betaine transporter Bta system is first dicovered in the betaine-synthesizing organisms.

博士
國立中興大學
生命科學系所
100
To adapt to the broad range of salt concentrations, methanogenic archaea accumulate potassium, α-glutamate, glycine betaine, β-glutamate, β-gultamine and Nε-acetyl-β-lysine as compatible solutes (osmolytes) to encounter the osmotic stress. The accumulation of Nε-acetyl-β-lysine as osmolyte is ubiquitous among methanogenic archaea, but not for other organisms. Nε-acetyl-β-lysine is synthesized from lysine through lysine 2,3-aminomutase (AblA) to form β-lysine and the acetyl group is further transferred to β-lysine by β-lysine acetyltransferase (AblB). The ablA and ablB genes were screened and obtained through PCR and Southern hybridization techniques from the marine Methanosarcina mazei N2M9705, halotolerant Methanocalculus chunghsingensis K1F9705bT and halophilic Methanohalophilus portucalensis FDF1T. The amino acid sequences of lysine 2,3-aminomutase (AblA) from these three methanogenic archaea contains ligands of the Fe/S cluster, SAM binding domain, PLP binding site and zinc binding sites; and β-lysine acetyltransferase (AblB) is a member of the GNAT [GCN5 (general control non-derepressible 5)-related N-acetyltransferase] superfamily of enzymes that contain R/Q-G/K-K/L-G-H/L-M/S-K/G segment. Compared the phylogenetic relationships of ablA and ablB genes along with 16S RNA genes, suggested the possible horizontal gene transfer may occur within halotolerant, halophilic methanogen and halophilic green-sulfur bacteria; or within Methanosarcina sp. and Bacillus/ Clostridium. Northern hybridization results showed that ablA and ablB were in one transcribed unit, and the osmolyte Nε-acetyl-β-lysine biosynthetic related genes from M. portucalensis FDF1T were salt up-regulated, but not immediately response to the temperature stress. The recombinant MpAblB and McAblB showed the high binding affinity to acetyl-CoA with Km value at 54.348 μM and 58.140 μM, respectively. Whereas the binding affinity to α-lysine were low with K0.5 value at 40~310 mM, suggested the AblB exihibits cooperative binding of α-lysine. Low affinity to α-lysine may due to the high specificity to β-lysine of the β-lysine acetyltransferase. The acetyltransferase activity of the recombinant MpAblB and McAblB were repressed by increasing level of potassium or sodium ions. Extremozymes from halotolerant and halophilic archaea were capable of salt-tolerant, solven-tolerant and retain catalytic activity in environments with low water activity. Lysine 2,3-aminomutase from methanoarchaea, especially from the halophilic methanogens, could act as potential biocatalysts for the synthesis of β-lysine, which can be applied in pharmaceutical applications.

碩士
國立中興大學
植物學系
89
Methanohalophilus portucalensis strain FDF1 can de novo synthesize betaine from glycine as compatible solutes while cells encounter the salt stress. The compatible solute betaine synthesizing through threefold of methylations from glycine was confirmed. Glycine N-methyltransferase (GNMT, EC 2.1.1.20) which transfer methyl group of S-adenosyl-L-methionine to glycine was purified by DEAE-Sephacel ion-exchange chromatography with the step gradient of potassium chloride and GNMT activity was detected at the elution with 0.5 M potassium. GNMT was a hexamer composed by 52 kD polypeptide. The purified GNMT composed 0.14 % of the total cell protein with specific activity of 0.56 nmol/mg×hr. Except using glycine as substrate, GNMT can also use sarcosine and dimethylglycine as substrates. The optimal assay conditions were performed under 0.4 mg GNMT, 1.15 mM SAM, 4 mM glycine and 400 mM KCl at 37℃. In addition, GNMT was strongly inhibited by the reaction product S-adenosylhomocysteine.

碩士
國立中興大學
生命科學系所
102
The halophilic methanoarchaeon Methanohalophilus portucalensis FDF1T can de novo synthesis the osmolyte betaine in response to extracellular salt stress, through three steps of methylation from glycine to form sarcosine, N, N-dimethylglycine and betaine by using glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine methyltransferase (SDMT). The level of post-translation was different with native GSMT and recombinant MpGSMT, and therefore it is of interest to purify native GSMT from FDF1T to compare their enzyme character and kinetics. To adapt to the broad range of salt concentrations, FDF1T could accumulate potassium ion (0.73M), and prone to be negative charge. Since the pI value of GSMT is 4.57, DEAE ion exchange was chosed for first line of protein purification. Further with Gel filtration and Centrifugal filter devices cut off the final 95% purity of GSMT from Methanohalophilus portucalensis FDF1T, were obtained and confirmed by Anti-MpGSMT made by recombinant MpGSMT. The specific methyltransferase activities of GSMT were 0.164 and 0.129 nmol/ μg/ hr on GMT and SMT, respectively. Moreover, the specific activities of GMT and SMT for the GSMT were significantly increased 4-30 fold under 2 M KCl and NaCl, and were remaining 30-40 % specific activities with 2 M glycine betaine.The dramatic activating effects of potassium and sodium ions on the glycine and sarcosine methyltransferase activities of GSMT, and the strong end product betaine inhibitory effect on GSMT suggested it is a key player in osmoregulation. On GSMT kinetic properties, the Km values for the GMT of GSMT at potassium levels of 0.5 and 1.5 M were 1.22 and 7.26 M, respectively, and for the SMT of GSMT were 1.0 and 0.35 M, respectively; the Vmax values for the GMT of GSMT at potassium ions conditons with 0.0128 and 0.512 nmol/ min, respectively, and for the SMT of GSMT were 0.0176 and 0.0243 nmol/ min. The affinity of GSMT and substrates were low. The affinity for GMT decreased and Vmax was considerably raised with an increasing level of K+, but MpGSMT was not. With these results demonstrated that activities are really difference with GSMT and MpGSMT.

碩士
國立中興大學
生命科學系所
94
Methanohalophilus portucalensis strain FDF1 can de novo synthesize glycine betaine as compatible solute to overcome the osmotic stress. The 13C-NMR and radiometric studies in extracts of M. portucalensis FDF1 proved that glycine betaine can de novo synthesize by transferring threefold methyl groups from S-adenosyl-L-methionine (SAM) to glycine. In this report, sarcosine dimethylglycine N-methyltransferase (SDMT) was purified from crude extract of FDF1 through DEAE-Sephacel ion exchange chromatography、Phenyl Sepharose hydrophobic interaction chromatography and Gel filtration Sephadex G-75. The specific methyltransferase activities of purified SDMT were 0.71 nmol/μg/min on sarcosine (SMT) and 3.06 nmol/μg/min on dimethylglycine (DMT). Analysis of 1 & 2-D gel electrophoresis and gel filtration suggests that SDMT was a monomer protein with the molecular weight of 33 KD and pI at 5.03. SDMT showed narrow substrate specificity with sarcosine and dimethylglycine only. On SDMT kinetic properties, the Km value of sarcosine and SAM were 2.29 mM and 0.21 mM respectively, the Vmax value was 0.83 nmol/μg/min and Kcat value was 0.44 (1/s) for SMT；the Km value of dimethylglycine and SAM were 3.76 mM and 0.59 mM respectively, the Vmax value was 4.88 nmol/μg/min and Kcat value was 2.68 (1/s) for DMT. The end product glycine betaine (2.0 M) did not effect the SMT activity but slightly repressed the DMT activity (with 65 % remains). On potassium and sodium effect, the SMT and DMT activity remains fairly constant with the addition of 200~1000 mM KCl or NaCl suggest that both potassium and sodium do not effect the SDMT activity.

碩士
國立中興大學
生命科學系
92
Methanohalophilus portucalensis strain FDF1 can de novo synthesize glycine betaine as compatible solute to encounter the osmotic stress. The 13C-NMR and radiometric studies in the extract of strain FDF1 proved that glycine betaine can de novo synthesize by transferring threefold methyl groups from S-adenosyl-L-met- hionine (SAM) to glycine. After DEAE-sephacel column separation the crude extract of strain FDF1 with step 0, 0.1, 0.2, 0.3, 0.4, 0.5 M KCl gradient, the sarcosine dimethylglycine N-methyltransferase (SDMT) that could transfer methyl group from SAM to sarcosine (SNMT) and dimethylglycine (DNMT) was detected at peak S2 eluted from 0.2 M KCl step gradient. Protein from the peak S2 showed SDMT activity at 800 mM KCl environment, the specific activity of SNMT was 2.49 nmol/μg•hr and the specific activity of DNMT was 9.69 nmol/μg•hr. Further purification the peak S2 with 50 kDa molecular weight cut off and sephadex G-100 column, significant SDMT activity was obtained at peak S2D-2 which composed by 23 kDa and 33 kDa polypeptide. The 23 kDa polypeptide can further separate into two polypeptides with pI value of 4.8 and 5.2 respectively by 2-D gel electrophoresis. The partial purified SDMT activities of peak S2D-2 was 5.33 nmol/μg•hr and 21.49 nmol/μg•hr for SNMT and DNMT, respectively. While grown at 12 % NaCl environment, cells of strain FDF1 accumulated 0.73 M KCl、0.31 M glycine betaine、0.46 M Nε-acetyl-β-lysine and 0.17 M β-glutamine as compatible solutes and the generation time was 13 hr. Base on the SDMT activity investigated in this study, under the condition of 800 mM KCl and sufficient substrate sarcosine, cells could synthesize 3.1 M required glycine betaine in 36 minutes.

博士
國立中正大學
化學暨生物化學研究所
101
In chapter 2-4 , the five-membered exocyclic DNA adducts are biologically very significant because of their potential to block DNA replication and transcription, induce DNA strand breaks, trigger, apoptosis, and cause gene mutations and chromosomal aberrations. These effects could lead directly to carcinogenesis. Common exocyclic DNA adducts, such as 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC), N2,3-ethenoguanine (N2,3-εG), and 1,N2-ethenoguanine (1,N2-εG), are responsible for blocking Watson-Crick base pairing. The present study provides useful information, such as geometric characteristics, electronic properties, and physical parameters, for the misincorporation of DNA nucleotides in the exocyclic adduct, based on different conformations and unique mutagenic properties that are essential for the continuous effort to understand the base-pairing specificity toward the determination of cancer etiology. From our results, it can be observed that the exocyclic DNA adducts readily paired with thymine which are in good agreement with the experimental observations. In chapter 5, the preferential interactions of glycine betaine (GB) with solvent components and the effect of solvent on its stability have been examined. In particular, the microsolvation of organic osmolyte and widely important osmoprotectant in nature as glycine betaine has been reported by using M06, MP2 and CCSD (T) method. A number of configurations of the clusters for one to seven water molecules have been considered for the microsolvation. Structures of stable conformers are obtained and denoted as b1a, b2a, b3a, b4a, b5a, b6a and b7a. It is observed from the interaction energy difference (ΔE) that only seven water molecules can be accommodated in the first solvation shell to stabilize GB. In chapter 6, DNA base pair A-T has been investigated by IR and NMR spectroscopy using DFT methods. The results have been analyzed in terms of infrared vibrational frequencies and 1H-NMR chemical shifts. Different types of interactions N-H…N, N-H…O and C-H…O types have been investigated in DNA base pairs. Although, previous reports argued about the third C-H…O type interaction in A-T base pair, such typical interaction has been analyzed thoroughly by IR and NMR spectroscopy using DFT methods. Our result shows that the CH...O interaction in the A-T base pair is a weak interaction compared to normal hydrogen bond interactions. In chapter 7, Ionic liquids are novel solvents of interest as greener alternatives to conventional organic solvents aimed at facilitating sustainable chemistry. As a consequence of their unusual physical properties, reusability, and eco-friendly nature, ionic liquids have attracted the attention of organic chemists. Numerous reports have revealed that many catalysts and reagents were supported in the ionic liquid phase, resulting in enhanced reactivity and selectivity in various important reaction transformations. However, synthetic chemists cannot ignore the stability data and intermolecular interactions, or even reactions that are directly applicable to organic reactions in ionic liquids. It is becoming evident from the increasing number of reports on use of ionic liquids as solvents, catalysts, and reagents in organic synthesis that they are not totally inert under many reaction conditions. While in some cases, their unexpected reactivity has proven fortuitous and in others, it is imperative that when selecting an ionic liquid for a particular synthetic application, attention must be paid to its compatibility with the reaction conditions. Even though, more than 200 room temperature ionic liquids are known, only a few reports have commented their effects on reaction mechanisms or rate/stability. Therefore, rather than attempting to give a comprehensive overview of ionic liquid chemistry, this review focuses on the non-innocent nature of ionic liquids, with a decided emphasis to clearly illuminate the ability of ionic liquids to affect the mechanistic aspects of some organic reactions thereby affecting and promoting the yield and selectivity.