Trimethylamine-n-oxide (TMAO) is a metabolite found in plasma/serum in humans. Elevated levels of TMAO have been associated with several types of heart disease. It’s therefore of interest to make a simple analytical method to analyse TMAO and other metabolites that are degraded to TMAO, including betaine. In this study, the goal was to develop a method for the sample preparation and analysis of these compounds in human plasma. Sample preparation was performed with an Ostro 96-well method for sample clean-up. The analysis was performed by ultraperformance liquid chromatography – hydrophilic interaction liquid chromatography – tandem masspectrometry (UPLC-HILIC-MS/MS) in multiple reaction monitoring (MRM)-mode using electrospray ionization in positive mode (ESI+)-mode as the ion source. The analytes eluted under five minutes and were all baseline separated in the chromatogram. TMAO and betaine were quantified in quality control (QC) plasma samples using external calibration. Concentration of TMAO ranged from 132 ng/mL – 253 ng/mL and 1025-2084 ng/mL for betaine. Due to the lack of isotopically labelled standards for TMAO and betaine, valine-d8 was tested as an internal standard for the extraction; however, it was not a suitable option due to the low recovery obtained (5-34%) and the low response in ESI+. The recovery needs to be investigated further using isotopically labelled TMAO or betaine. Overall, the developed UPLC-HILIC-MS/MS method was found to be suitable for analysis of TMAO and betaine in human plasma. Further development and validation is required before application to samples from clinical studies.

<p style="text-align: justify;">Betain je bioaktivno jedinjenje, koje ima značajne fiziolo&scaron;ke funkcije u čovekovom organizmu kao osmolit i donor metil grupa za mnoge biohemijske procese. Endogena sinteza betaina je uglavnom nedovoljna da zadovolji potrebe organizma te je zbog toga neophodan njegov unos preko hrane. Žitarice predstavljaju glavni izvor betaina u ljudskoj ishrani. Jedan od načina da se poveća unos betaina u ljudskoj ishrani je preko funkcionalne hrane obogaćene betainom. Prema regulativi evropskog udruženja cerealija za doručak (European Breakfast Cereal Association) iz 2012. god. (Commission Regulation (EU) No 432/2012) dozvoljeno je deklarisanje zdravstvene izjave za prehrambene proizvode koji sadrže najmanje 500 mg betaina po porciji (40g flips proizvoda) uz navođenje napomene da se zdravstveni efekti mogu očekivati ukoliko se dnevno unese 1500 mg betaina. Zdravstvena izjava glasi: &bdquo;betain doprinosi normalnom metabolizmu homocisteina&ldquo;. Povećano interesovanje za zdravu ishranu dovelo je do veće potražnje za alternativnim žitaricama, gde spelta zbog svojih biolo&scaron;kih, agronomskih, prehrambenih i medicinskih karakteristika kao i bogatih nutritivnih svojstava, zauzima značajno mesto. Glavni cilj ove teze je bila optimizacija ekstrudiranja i proizvodnje funkcionalnog flips proizvoda od speltinog integralnog bra&scaron;na sa dodatkom betaina na dvopužnom ekstruderu. Prvi put je u procesu prehrambenog ekstrudiranja primenjena analiza vi&scaron;eciljne optimizacije u kombinaciji sa neuronskim mrežama i genetskim algoritmom kako bi se dobio maksimalan sadržaj betaina u flips proizvodu pri minimalnoj potro&scaron;nji specifične mehaničke energije. Definisan je nov funkcionalni proizvod sa betainom koji bi potencijalno pro&scaron;irio asortiman flips proizvoda na trži&scaron;tu i utvrđene su njegove fizičke, funkcionalne, teksturalne, reolo&scaron;ke i senzorske osobine. Sadržaj betaina i potro&scaron;nja specifične mehaničke energije su ključni izlazi koji predstavljaju konkurentne ciljeve u procesu ekstrudiranja. Za proizvodnju flips proizvoda pobolj&scaron;anog nutritivnog kvaliteta i funkcionalnog profila koji sadrži od 1601,6 do 1764,7 mg betaina u 40 g (čime se i zadovoljava preporučeni dnevni unos betaina) i kome odgovara potro&scaron;nja specifične mehaničke energije od 97,4 do 114,1 Wh/kg optimalni su sledeći procesni parametri: protok materijala od 20,45 do 24,04 kg/h, vlaga materijala od 18,6 do 19,44 % i brzina obrtanja pužnice od 250 obrtaja u minuti. Da bi se dobili pouzdani rezultati za sadržaj betaina u spelti i flips proizvodima razvijena je, optimizovana i validovana metoda tečne hromatografije visokih perfomansi. Metoda je uspe&scaron;no primenjena i u analizi betaina u žitaricama, pseudožitaricama i njihovim proizvodima.</p> / <p>Betaine is a bioactive compound that has significant physiological functions in the human organism as an osmolite and methyl group donor for many biochemical processes. The endogenous synthesis of betaine is generally insufficient to satisfy the requirements of the organism, therefore its intake is necessary through the food. Cereals are the main sources of betaine in human nutrition. One of the ways to increase betaine intake in human nutrition is through betaine enriched functional foods. In the 2012 European Union Regulation (Commission Regulation (EU) No 432/2012), it is permissible to declare a health statement for foods containing at least 500 mg betaine per portion (40g of extruded product), stating that the health effects can be expected if 1500 mg of betaine are consumed on a daily basis. The health statement states: &quot;Betaine contributes to the normal metabolism of homocysteine.&quot; Increased interest in healthy food has led to the higher demand for alternative cereals, where spelt takes a significant place due to its biological, agronomic, nutritional and medical characteristics, as well as its rich nutritional properties. The main goal of this work was the optimization of extrusion and production of the functional snack product from spelt wholegrain flour with the addition of betaine which is performed on twin-screw extruder. According to our best knowledge, there was no investigation in which the multi-objective optimization (MOO) analysis coupled with artificial neural network and genetic algorithm was applied to the extrusion cooking process in order to achieve simultaneously maximum betaine content and minimum energy consumption in the snack spelt product. A new functional snack product that can potentially expand the range of snack products and its physical, functional, textural, sensory and rheological properties were defined. The betaine content and specific mechanic energy are the key outputs that represent competing objectives in the extrusion proces. To produce snack product with improved nutritive quality and functional profile which contains betaine content from 1601,6 to 1764,7 mg/40g, which would contribute to the recommended daily intake of betaine, correspond to specific mechanical energy from 97,4 to 114,1 Wh/kg, following optimal parameters were obtained: feed rate from 20,45 to 24,04 kg/h, moisture content from 18,6 to 19,44 % and screw speed at 250 rpm. Also, optimization, development and validation of the high pressure liquid chromatographic method for measuring betaine content in spelt and snack products was also designed and applied.</p>

Seasonally high nighttime temperatures (HNT) along the United States Gulf Coast and in regions of similar climate, during the critical stages of development, could reduce rice yield and quality. To study the effects of HNT on plant physiology, a method for applying a controlled heating treatment to plant canopies was developed using overhead infrared heaters, which are relatively inexpensive and are accurate, precise and reliable in rapidly controlling the temperature. The apparatus successfully maintained air temperatures within the set points plus/minus 0.5 degrees C, and was used for all the experiments. Several experiments were conducted to determine the response of various physiological parameters during and following exposure of rice plants to HNT (32 degrees C) or ambient nighttime temperature (ANT) (27 degrees C) starting from 2000 h until 0600 h, and with or without plant growth regulator treatments. The plant growth regulator treatments included alpha-tocopherol (vitamin E), glycine betaine (GB), and salicylic acid (SA), which play different roles in inducing thermo-tolerance in plants. High nighttime temperature had no effect on plant height, number of tillers and panicles, or rice net leaf photosynthetic rates. However, HNT increased leaf respiration (dark respiration in the night) (21%) and decreased membrane thermo-stability (60%), pollen germination (20%), spikelet fertility (18% as a % of total spikelets), grain length (2%), and grain width (2%). The HNT also hastened plant development. The combinations of these effects decreased rice yield by 90%. Moreover, under HNT, there were decreases in leaf chlorophyll concentration (7%) and nitrogen concentration (18%). Application of GB and SA increased total antioxidant capacity of the rice plants by 17%, thereby decreasing the leaf respiration rates, increasing membrane thermo-stability, pollen germination, and spikelet fertility, thus increasing the yield. High nighttime temperature decreased leaf starch concentration (14%), grain total nonstructural carbohydrate (TNC) concentration (9%), and grain extractable invertase activity (20%). Vitamin E- or GB-treated plants had greater grain soluble-sugar concentrations, whereas SA-treated plants had greater leaf soluble-sugar concentrations and lower grain TNC concentrations. Invertase activity was shown to be not rate limiting or required for sucrose degradation for starch synthesis in grain of 'Cocodrie' rice under short-term high nighttime temperatures exposures during grain filling. In conclusion, HNT decreased rice yield by increasing plant respiration, rate of crop development, and decreasing membrane thermo-stability, pollen germination, spikelet fertility and grain dimensions. Exogenous application of GB and SA increased yields under HNT, possibly acting through increased antioxidant levels, which might have protected the membranes and enzymes against heat-induced ROS-mediated degradation.

This dissertation evaluated nutritional approaches such as the addition of betaine, prebiotics, probiotics, transitional metabolic substrates, and β-mercaptoacetate (MAA; a compound which inhibits β-oxidation) to the diet of lactating dairy cows to determine their impact on physiological, metabolic, hormonal and production responses during thermal stress. The first objective was to evaluate the use of an organic osmolyte, betaine to reduce the impact of heat stress (HS). Cows were fed either 0 (control; CON), 57 mg/kg BW (mid) or 114 mg/kg (high; HI) body weight (BW) betaine and subjected to thermoneutral (TN) and HS conditions. There was an increase in milk yield during TN with HI betaine over controls (P< 0.01), but the advantage was lost during HS. Plasma glucose increased during HS in HI dose cows compared to control (P < 0.01) as did plasma insulin (P = 0.01). Betaine increased milk production during TN and plasma glucose in HS, but did not improve the HS response. Objective two evaluated the use of a probiotic or direct fed microbial (DFM), Calsporin (Bacillus subtilus C-3102) to decrease the effects of HS in dairy cows. We hypothesized that feeding Calsporin prior to and during HS would reduce pathogenic strains of bacteria, maintain commensal microbes, and improve ruminal anaerobic fermentation resulting in improved milk yield (MY). Milk yield was numerically increased (1.26 kg, P = 0.11) in cows fed Calsporin during TN but was reduced under HS (-2.67 kg, P < 0.01) and milk protein content was decreased (P = 0.05). The DFM tended to decrease somatic cell count (SCC) across periods (P = 0.07). Calsporin addition to the diet did not affect respiration rates and was associated with higher rectal temperature at 1800 in HS (P = 0.02). The expression of heat shock protein 27 (HSP27) was decreased with Calsporin treatment (P = 0.03) and in both HS and TN. The fecal microbial count did not change with the exception of the Calsporin strain in treated animals (P < 0.01). The third objective was to feed OmniGen-AF (OG) to dairy cows before and during thermal stress. We hypothesized that feeding OG to HS dairy cows will improve the immune response, and decrease production losses associated with HS. Cows fed OG maintained lower SCC compared to control (P < 0.01) during the recovery period. We did not detect differences between groups in serum calcium while serum non-esterified fatty acid (NEFA) concentrations (P = 0.10) tended to be greater in OG fed cows across the Agricultural Research Center (ARC) portion including HS. Serum Adrenocorticotropic hormone (ACTH) levels were greater in OG cows (P<0.0001) across all sample days. Feeding OG reduced the HS response including serum Cortisol. The final study measured the effects of the metabolic substrate β-hydroxybutyrate (BHB) during HS on feed intake and metabolites. Under TN conditions the cows received a bolus dose of BHB and dry matter intake (DMI) and metabolites were measured. The second part of this study used a bolus of MAA to limit the up-stream production of acetyl-CoA available for ketogenesis by inhibiting ß-oxidation. We proposed that dosing lactating dairy cows with BHB would decrease DMI, increase plasma insulin, decrease NEFAs and increase skin temperature by vasodilatation. The same cows were then subjected to HS and dosed with saline and MAA on different test days. The infusion of BHB increased skin temperature (time 0.5, 1, 2, 3 and 4°C r² =0.98 with serum BHB) and decreased serum NEFA levels (P < 0.01). There was no change in mean DMI, glucose or insulin. The bolus of MAA decreased feed intake, vaginal temperature, and insulin. There was an increase in serum BHB with the initial dose of MAA and an initial decrease in serum glucose (P < 0.0001) with MAA. Serum glucose increased as insulin decreased with MAA. The infusion of BHB did not alter feed intake in this study despite high plasma levels of BHB.

This work deals with the synthesis and photophysical study of fluorescent molecules, aiming to construct new chemical sensors for the selective detection of environmentally or biologically important metal ions. A series of fluoroionophores consisting of triazole moiety and different fluorophores was utilized to investigate the functions of triazole group in chemosensors. Our comprehensively photophysical and complexing studies of these ligands demonstrated that triazole groups could contribute as part of conjugated fluorophores, as metal binding sites or linkers. Several selective chemosensors were successively developed for the detection of Cu2+ or Hg2+. By incorporating dansyl fluorophore into calix[4]bisazacrown, a new potassium chemosensor was constructed. A sulfonate fluorescent calix[4]bisazacrown was created for the selective and ratiometric detection of Al3+ in lutidine buffer solution at pH 6.0 with a satisfying sensitivity. Finally, we aim to realize modulation of cation binding through the photoinduced charge redistribution in the excited state. Two binding motifs based on fullerene and betaine pyridinium were designed for this purpose. Preliminary results demonstrated that PET in fullerene might be not suitable but pydinium betaine hold great potential in the cation translocations.

Arthrobacter sp. yra dirvoje paplitusios bakterijos, kurios gali prisitaikyti prie nepalankių aplinkos sąlygų, tokių kaip osmosinis šokas ar maisto medžiagų trūkumas. Osmoreguliacijoje daugelis organizmų naudoja glicino betainą. Yra žinoma, kad Arthrobacter globiformis bakterijos gali panaudoti glicino betainą kaip anglies ir azoto šaltinį, tačiau nebuvo aišku, ar Arthrobacter globiformis gali šią medžiagą naudoti kaip osmoprotektorių. Iš Arthrobacter globiformis buvo klonuotas DNR fragmentas, kuriame buvo identifikuoti genai, kurie tiesiogiai ir netiesiogiai dalyvauja glicino betaino katabolizme. Struktūrinė genų organizacija leidžia manyti, kad šios bakterijos gali panaudoti glicino betainą kaip osmoprotektorių, Taip pat buvo įdomu patikrinti, ar tokią funkciją glicino betainas gali vykdyti ir kitose A. globiformis giminingose bakterijose. Tyrimui buvo pasirinkti tipiniai Arthrobacter genties bakterijų kamienai (A. atrocyaneus, A. citreus, A. crystalopoietes, A. globiformis, A. ramosus, A. sulfureus bei Arthrobacter spp. (KA3, P3, KA2V2, PY22, KA2, GAZ21, P2G, KA4, KA2V3, GAZ3, PRH1, PY21, VM22, VP23, RD1, VM22, VP22, VP3, VPW7, VPS4, 96, 94, 85, 68M, 83 68B, BL-3 ir 1-IN). Darbo metu paaiškėjo, kad šį junginį apsaugai nuo osmosinio streso naudoja tik dalis Arthrobacter genties bakterijų. Glicino betainas artrobakterėse dažniau naudojamas kaip anglies šaltinis. / Arthrobacter spp. are wide-spread soil bacteria, which are adapted to grow under osmotic stress and shortage in available carbon (energy) sources. Many organisms utilise glycine betaine as an osmoprotectant. It is known, that Arthrobacter globiformis can use glycine betaine as a sole carbon and nitrogen source, however, it is unclear whether this bacterium use this substrate as osmoprotectant. A DNA fragment harbouring genes, which directly and indirectly are involved in glycine betaine degradation, was cloned from A. globiformis. The structural organization of these genes suggested, that this bacterium could use glycine betaine as an osmoprotectant. So, it was tested if other Arthrobacter spp. strains use glycine betaine to protect from osmotic stress. Typical Arthrobacter sp. strains (A. atrocyaneus, A. citreus, A. crystalopoietes, A. globiformis, A. ramosus, A. sulfureus and Arthrobacter spp. (KA3, P3, KA2V2, PY22, KA2, GAZ21, P2G, KA4, KA2V3, GAZ3, PRH1, PY21, VM22, VP23, RD1, VM22, VP22, VP3, VPW7, VPS4, 96, 94, 85, 68M, 83 68B, BL-3 and 1-IN) were chosen in this study. It turned out, that less than a half of studied strains use glycine betaine as osmoprotectant. The arthrobacters used glycine betaine more as a sole carbon source than as an osmoprotectant.

Thesis--University of Turku, Finland, 2003. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis--University of Turku, Finland, 2003. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Made available in DSpace on 2015-04-01T14:16:04Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1756619 bytes, checksum: d052f1be18e53f2beddaa57cda5b0a22 (MD5) Previous issue date: 2012-12-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Substrate-binding subunits are very important components of the solute importation system, known as the osmoprotectant system, which consists of a membrane protein belonging to the ABC superfamily. These molecules recognize specific substrates that have different physiological roles in prokaryotes, i.e. roles that contribute to the survival of these organisms in environments with high concentrations of salt. Using MEGA 5.05 software, this study performed a phylogenetic analysis of 431 nucleotide sequences of these subunits, orthologous to each other, collected from the database contained on the website http://www.genome.jp/kegg/. As a result of this analysis, phylogenetic trees were generated that clearly demonstrated that there was a horizontal transfer of some genes due to the sharing by different organisms. Also, two probable ancestral sequences were generated that showed homology with permeases that transport choline, glycine betaine and carnitine, which are trimethylamines currently present in various prokaryotes. Therefore, this system probably arose in prokaryotic organisms with the basic function of capturing nutrients, and by performing this basal function of being shared with other organisms, was fixed to the genome. However, because of the diversification of habitats by the prokaryotes, this system contributed decisively to the adaptation of these organisms to different environments, especially environments that had a high salt concentration; thus, acting and being currently characterized as a system of osmoprotection. / As subunidades ligadoras de substrato são componentes muito importantes do sistema de importação de soluto conhecido, como sistema de osmoproteção, que consiste em uma proteína de membrana pertencente à superfamlía ABC. Estas moléculas reconhecem substratos específicos que apresentam papéis fisiológicos diversos em procariotos, incluindo a colaboração para a sobrevivência destes organismos em ambientes com elevada concentração de sal. Utilizando o software MEGA 5.05, foi realizada uma análise filogenética de 431 sequências nucleotídicas destas subunidades, ortólogas entre si, coletadas a partir do banco de dados contido no site ttp://www.genome.jp/kegg/. Como resultado desta análise, foram geradas árvores filogenéticas que demonstraram claramente que houve a transferência horizontal de alguns dos genes devido ao ompartilhamento por organismos diferentes. Foram geradas também duas prováveis sequências ancestrais que apresentam homologia com permeases que transportam colina, glicina betaína e carnitina, que são aminas trimetiladas, presentes atualmente em diversos procariotos. Portanto, este sistema provavelmente surgiu em organismos procarióticos com a função básica de captura de nutrientes e por desempenhar esta função basal, ao ser compartilhado com outros organismos, foi fixado aos genomas. No entanto, a partir da diversificação de habitats, por parte dos procariotos, este sistema colaborou de forma decisiva para a adaptação destes organismos aos mais diversos ambientes, incluindo, especialmente os ambientes que apresentavam uma elevada concentração de sal, atuando e sendo caracterizado atualmente como um sistema de osmoproteção.

Ce travail porte sur la synthèse et l'étude photophysique des molécules fluorescentes, visant à construire de nouveaux capteurs chimiques pour la détection sélective des ions métalliques importants en biologie ou pour l’environnement. Une série de fluoroionophores contenant le motif triazole et différents fluorophores ont été utilisés pour étudier les fonctions du groupe triazole dans les senseurs chimiques. Nos études photophysiques de ces ligands complexants démontrent que le triazole pourrait contribuer dans le cadre de fluorophores conjugués, comme les sites de complexation de métal ou de linkers. Plusieurs senseurs chimiques sélectifs ont été successivement mis au point pour la détection du Cu2+ ou du Hg2+. En incorporant le fluorophore dansyle sur calix[4]bisazacrown, une nouvelle capteur chimique de potassium a été construit. Un sulfonate fluorescente calix [4] bisazacrown a été conçu et synthétisé pour la détection sélective et ratiométrique de l’aluminium dans une solution tampon de lutidine à pH 6,0 avec une sensibilité satisfaisante. Enfin, nous avon cherché à réaliser une modulation de la complexation de cation par la redistribution de charge photoinduite à l'état excité. Deux motifs de liaison sur la base de fullerène et de pyridinium bétaïne ont été conçus à cet effet. Les résultats préliminaires ont mis en évidence un effet de type PET dans les composés à base de fullerène et les composés à base de pyridinium betaine sont trés prometteur dans la translocation de cation. / This work deals with the synthesis and photophysical study of fluorescent molecules, aiming to construct new chemical sensors for the selective detection of environmentally or biologically important metal ions. A series of fluoroionophores consisting of triazole moiety and different fluorophores was utilized to investigate the functions of triazole group in chemosensors. Our comprehensively photophysical and complexing studies of these ligands demonstrated that triazole groups could contribute as part of conjugated fluorophores, as metal binding sites or linkers. Several selective chemosensors were successively developed for the detection of Cu2+ or Hg2+. By incorporating dansyl fluorophore into calix[4]bisazacrown, a new potassium chemosensor was constructed. A sulfonate fluorescent calix[4]bisazacrown was created for the selective and ratiometric detection of Al3+ in lutidine buffer solution at pH 6.0 with a satisfying sensitivity. Finally, we aim to realize modulation of cation binding through the photoinduced charge redistribution in the excited state. Two binding motifs based on fullerene and betaine pyridinium were designed for this purpose. Preliminary results demonstrated that PET in fullerene might be not suitable but pydinium betaine hold great potential in the cation translocations.