The prevalence of cardiovascular disease is a major public health concern worldwide. It has been theorized that diets rich in fruits and vegetables may be protective against the development of cardiovascular disease mainly through their high content of flavonoids. Flavonoids were thought to influence traditional risk factors of cardiovascular disease such as blood pressure, lipid profile, and systemic inflammation. Recent clinical studies have shown that this may not be the case. The production of trimethylamine oxide (TMAO) by the gut microbiota from dietary sources of choline has been associated with an increased risk of cardiovascular events. The objectives of this study were to determine the effects of a high flavonoid diet on gut microbiota composition and plasma trimethylamine oxide concentrations. Potential benefits of this research include the determination of a potential correlation between diet and markers of traditional and non-traditional risk factors for cardiovascular disease. Also, the effects that a high flavonoid diet has on the composition of the gut microbiota and plasma trimethylamine oxide concentrations may provide insight into possible dietary interventions to prevent cardiovascular disease.

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There is an association between the human microbiome and disease. Gut microbes metabolize dietary sources to release trimethylamine (TMA). TMA is absorbed and then oxidized by flavin monooxygenase 3 (FMO3) to form trimethylamine N-oxide (TMAO). Elevated TMAO is associated with increased risk of cardiovascular disease and type 2 diabetes; however, the causal nature is unclear. There is also limited evidence supporting the efficacy of strategies to reduce accumulation of TMAO. Therefore, the purpose of these studies is to determine the effects of increases in TMAO on cardiometabolic health. In study 1, healthy sedentary and endurance trained males consumed a high fat diet. Blood samples were obtained in a fasted state and every hour during a 4-hour high fat challenge. We hypothesized sedentary individuals would produce higher TMAO concentrations. In study 2, healthy sedentary individuals consumed an acute 1000 mg dose of choline (CHOL) and placebo (PLC). Fasted blood samples were collected, flow-mediated dilation (FMD) and oral glucose tolerance (OGT) were measured. In study 3, healthy sedentary individuals consumed 4-wks of CHOL and PLC. Fasted blood samples were collected, FMD and OGT were measured. We hypothesized acute and 4-wk choline supplementation would impair FMD and OGT. In study 1, neither fasting (1.49± 1.2 µM vs. 2.25 ± 1.4 µM, p>0.05) or postprandial TMAO changed significantly with the HFD in sedentary or endurance trained individuals even with the endurance group consuming more TMA dietary precursors. Study 2 found increased plasma TMAO concentrations after choline supplementation on day 1(PLC; 4.14 ± 2.6 μM vs. CHOL; 23.6 ± 33.8 μM, p=0.018) and day 2 (PLC; 5.13±4.9 μM vs. CHOL; 32.6±37.5 μM, p=0.082) however, there were no differences in OGT or FMD. Study 3 found no differences in FMD or OGT following 4-wks of choline consumption. In summary, there were no differences between sedentary and endurance trained individuals fasting or post-prandial TMAO. There was also no effect on acute or 4-wk supplementation of choline on FMD and OGT. More research is needed to understand effects of elevated TMAO on cardiometabolic health. / Doctor of Philosophy / For years, research has been performed to identify the health effects of eating large amounts of red meat on cardiovascular disease (CVD). Consuming red meat, fish, poultry and eggs increases a substance created during digestion and metabolism, called trimethylamine N-oxide (TMAO). Elevated TMAO has been associated with increased risk of CVD and type 2 diabetes but the direct causes are unknown. The purpose of these studies is to determine the effects of increases in TMAO on health in humans. Study 1 included healthy, sedentary and endurance trained males who consumed a high fat diet. Blood samples were collected to measure TMAO before and after a high fat meal. Study 2 included healthy, sedentary males and females who consumed 2 days of 1000 mg of choline, which is commonly found in red meat fish and eggs, and a placebo (carbohydrate) after subjects completed a series of tests to evaluate health. Study three included healthy, sedentary males and females who consumed 4-weeks of 1000 mg of choline per day and a placebo (carbohydrate). Following supplementation subjects underwent a series of tests to assess health. Overall, there were no differences found between sedentary and endurance trained individuals. Acute and 4-week supplementation of choline did not affect measures of blood sugar or blood vessel function.

Trimethylamine, (CH3)3N, (TMA), odors are often associated with limed and polymer conditioned sludges. This odor has a fishy smell and can be a nuisance to the community surrounding a wastewater treatment plant or land application site. Several factors are thought to determine the amount of TMA generated from limed biosolids. These are, the presence of cationic polymer, the polymer dose, the time between addition of polymer and lime stabilization, shear imparted on the sludge in the dewatering process and dewatered cake solids concentration. All of these were investigated in this study. The results showed that TMA could be generated from sludge that did not contain polymer but the concentrations were low compared to sludge conditioned with cationic polymer. As the polymer dose increased, the TMA increased. Shear also showed to play an important role for TMA production. In addition to higher shear increasing the polymer demand, shear in itself can increase TMA generation. However, the most important factor in generating TMA was the time between conditioning and liming. If this time was minimized, little TMA was produced, even at high polymer doses. Data also suggests that methanogens play an important role in the breakdown of TMA. / Master of Science

Trimethylamine N-oxide (TMAO) is a metabolite derived from dietary choline, betaine, and carnitine via intestinal microbiota metabolism. In several recent studies, TMAO has been shown to directly induce inflammation and reactive oxygen species (ROS) generation in numerous cell types, resulting in cell dysfunction. However, whether TMAO will impact stem cell properties remains unknown. This project aims to explore the potential impact of TMAO on mouse embryonic stem cells (mESCs), which serve as an in vitro model of the early embryo and of other potent stem cell types. Briefly, mESCs were cultured in the absence (0mM) or presence of TMAO under two different sets of treatment conditions: long-term (21 days), low-dose (20µM, 200µM, and 1000µM) treatment or short-term (5 days), high-dose (5mM, 10mM, 15mM) treatment. Under these treatment conditions, mESC viability, proliferation, and stemness were analyzed. mESC properties were not negatively impacted under long-term, low-dose TMAO treatment; however, short-term, high-dose treatment resulted in significant reduction of mESC viability and proliferation. Additionally, mESC stemness was significantly reduced when high-dose treatment was extended to 21 days. To investigate an underlying cause for TMAO-induced loss in mESC stemness, metabolic activity of the mESCs under short-term, high-dose TMAO treatment was measured with a Seahorse XFe96 Analyzer. TMAO treatment significantly decreased the rate of glycolysis, and it increased the rate of compensatory glycolysis upon inhibition of oxidative phosphorylation (OxPHOS). It also significantly increased the rate of OxPHOS, maximal respiratory capacity, and respiratory reserve. These findings indicate that TMAO induced a metabolic switch of mESCs from high glycolytic activity to greater OxPHOS activity to promote mESC differentiation. Additionally, TMAO resulted in increased proton leak, indicating increased oxidative stress, and elucidating a potential underlying mechanism for TMAO-induced loss in mESC stemness. Altogether, these findings indicate that TMAO decreases stem cell potency potentially via modulation of metabolic activity. / Master of Science / Trimethylamine N-oxide (TMAO) is a metabolite that is produced by the bacteria in the gut after the consumption of specific dietary ingredients (e.g., choline, carnitine, betaine). These ingredients are commonly found in meat and dairy products, and thus make up a large part of the average American diet. Recently, it was discovered that high TMAO levels in the bloodstream put people at an increased risk for heart disease, neurodegenerative diseases (e.g., Alzheimer's Disease), diabetes, stroke, and chronic kidney disease. At the cellular level, there is evidence that TMAO increases inflammation and the production of oxygen radicals, which causes cells to lose their function and promotes the onset of disease. TMAO has been well studied in adult cell types; however, no one has investigated whether TMAO will impact cells of the early embryo. This project aims to explore the impact of TMAO on mouse embryonic stem cells (mESCs), which are cells that represent the early stage of embryonic development and are critical for proper development of the final offspring. In addition, mESCs may also help to provide insight into how TMAO impacts other stem cell types, some of which are present throughout the entire human lifespan and play an important role in the body's ability to repair itself and maintain overall health. My project demonstrated that TMAO does not impact the overall health of mESCs under normal conditions, which signifies that TMAO generated by a pregnant mother may not directly impact the early embryonic stage of development. Further studies should be conducted to determine the potential impact of TMAO on late stages of embryonic and fetal development. Next, to simulate diseased conditions, the mESCs were treated with extremely high concentrations of TMAO in order to determine what concentration of TMAO will negatively impact these cells. It was found that at 5mM TMAO, mESCs begin to lose their basic properties and become dysfunctional. They are impaired in their viability, growth, ability to become other cell types, and in their metabolic activity. These mESC properties are shared with several types of adult stem cells, and therefore, these findings help to provide insight into how TMAO may impact stem cells found in the adult body which are exposed to a lifetime of high TMAO levels. In the future, we would like to further explore the impact of TMAO on mESCs at the molecular level as well as examine the direct impact of TMAO on other stem cell types.

Orientadores: Pedro Eduardo de Felicio, Jana Pickova / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-04T20:04:37Z (GMT). No. of bitstreams: 1 LapaGuimaraes_Judite_D.pdf: 14813702 bytes, checksum: ceaf92aa9f32ece3f3b4c5ef620aa4ee (MD5) Previous issue date: 2005 / Resumo: Em função da alta perecibilidade do pescado, a avaliação do frescor e qualidade deste alimento deve ser precisa e baseada em métodos consolidados. Bases nitrogenadas voláteis (BNV), trimetilamina (TMA), aminas biogênicas, nucleotídeos, uréia e triptofano livre têm sido propostos como índices de frescor para pescado e podem ser efetivos ou não dependendo da espécie de pescado, microbiota contaminante e condições de armazenamento. A quantificação dos teores de aminas biogênicas é também importante devido ao seu potencial tóxico. Os objetivos deste estudo foram avaliar a utilidade das aminas biogênicas, BNV, TMA, triptofano livre e uréia como índices de qualidade e frescor em peixes e moluscos cefalópodes, desenvolver e avaliar sistemas de solventes para separação de aminas biogênicas por cromatografia de camada delgada e avaliar o efeito do tempo e temperatura de derivatização sobre a conversão das aminas biogênicas em seus derivados dansilados. Derivados dansilados de agmatina (AGM), putrescina (PUT), triptamina (TRY), cadaverina (CAD), histamina (HIS), espermidina (SPD), espermina (SPM), tiramina (TYR) e feniletilamina (PHE) foram separados utilizando-se o sistema de solventes clorofórmio: éter dietílico: trietilamina (6:4:1 - v/v) seguido de clorofórmio: trietilamina (6:1 ¿ v/v). A melhor condição para derivatização das aminas biogênicas com cloreto de dansila foi 1h a 40oC. AGM permaneceu no local de aplicação (RF=0,0), o que indica que a metodologia deve ser utilizada com cautela para a determinação desta amina. As percentagens de recuperação de TRY, SPM, SPD e TYR foram baixas, indicando que a metodologia para extração destas aminas deve ser aperfeiçoada. BNV e TMA foram consideradas índices de frescor inadequados para bacalhau (Gadus morhua) e hadoque (Melanogrammus aeglefinus). O padrão de produção mais acentuado a partir da segunda semana de armazenamento, caracterizou as aminas voláteis e as aminas biogênicas CAD e PUT como índices de deterioração para bacalhau e hadoque. Uréia se mostrou inadequada como índice de qualidade para as duas espécies de pescado estudadas. Modelos de regressão linear e quadrática indicaram um aumento progressivo dos teores de AGM em lula (Illex coindetii), de uréia em sépia (Sepia officinalis), e de BNV e triptofano livre nas duas espécies de cefalópodes desde o início do armazenamento. BNV e triptofano livre foram considerados bons índices de frescor para I. coindetii e S. officinalis, pois seus teores aumentaram significativamente (p<0,05) desde o início do armazenamento. AGM e uréia foram consideradas índices de deterioração para I. coindetii e S. officinalis, respectivamente. Os teores de TMA (% NNP) aumentaram significativamente (p<0,05) durante a primeira semana de armazenamento em I. coindetii o que sugere sua utilidade como índice de frescor para lula. Para S. officinalis TMA é mais adequada como índice de deterioração / Abstract: Fish and shellfish muscle is highly susceptible to spoilage during storage. Because quality quickly decreases during storage reliable chemical indices for quality and freshness evaluation are greatly needed. Total volatile bases-nitrogen (TVB-N), trimethylamine (TMA), biogenic amines, nucleotides, urea and free tryptophan have been suggested as freshness indices for fish and shellfish. Such indices are useful depending on the species, microbial flora and storage conditions. Biogenic amines levels are also cause of concernment due to their toxicological effects. The objectives of this study were to evaluate the usefulness of biogenic amines, TVB-N, TMA, free tryptophan and urea as freshness and quality indices for fish and cephalopods; to develop and evaluate solvent systems for biogenic amines separation by thin-layer chromatography; and to evaluate the effects of time and temperature of derivatisation on the conversion of biogenic amines to their dansyl derivatives. Dansyl derivatives of agmatine (AGM), putrescine (PUT), tryptamine (TRY), cadaverine (CAD), histamine (HIS), spermidine (SPD), spermine (SPM), tiramine (TYR) and phenylethylamine (PHE) were separated using the solvent system chloroform: diethyl ether: triethylamine (6:4:1 ¿ v/v), followed by chloroform: triethylamine (6:1 ¿ v/v). The best dansylation condition was 1h at 40oC. AGM remained at the start position, indicating the determination of AGM by this method should be considered with caution. The percentages of recovery of TRY, SPM, SPD and TYR were low, indicating that the extraction methodology must be improved. TVB-N and TMA were considered inappropriate as freshness indices for cod (Gadus morhua) and haddock (Melanogrammus aeglefinus). CAD and PUT showed the greatest increases during the storage, but their levels did not increase significantly (p<0.05) during the first week. Volatile amines and biogenic amines were characterized as spoilage indices for G. morhua and M. aeglefinus due to the intense increasing of their amounts from the second week of storage. Urea was not useful as a quality index for cod and haddock. Linear and quadratic models indicated progressive increasing of AGM levels in squid (Illex coindetii), urea in cuttlefish (Sepia officinalis) and also increasing of TVB-N and free tryptophan in both cephalopods, since the beginning of the storage. TVB-N and free tryptophan were considered good freshness indices for both cephalopod species, because their levels significantly increased (p<0.05) during the first week of storage. AGM and urea were useful as spoilage indices for I. coindetii and S. officinalis, respectively. It was observed a significant increase of TMA (% of NNP) during the first week of storage in I. coindetii indicating the TMA usefulness as freshness index for squid. For S. officinalis TMA was a good spoilage index / Doutorado / Doutor em Tecnologia de Alimentos

Microbiome-derived trimethylamine N-oxide (TMAO) has been associated with platelet hyperreactivity and subsequent atherogenesis. Whether physiological TMAO-levels influence plateletderived lipid mediators remains unknown. Little is known about pre-analytic factors potentially influencing TMAO concentrations. We aimed at developing a quantitative LC-MS/MS method to investigate in-vivo and in-vitro pre-analytical factors in TMAO analysis to properly assess the proposed activating effect of TMAO on platelets. TMAO, betaine, carnitine, and choline were analyzed by HILIC-ESI-MS/MS within 6 min total run time. Method validation included investigation of reproducibility, recovery, sensitivity, and in-vitro pre-analytical factors. A 24-h monitoring experiment was performed, evaluating in-vivo pre-analytical factors like daytime or diet. Finally, the effects of different TMAO concentrations on platelet activation and corresponding alterations of plateletderived eicosanoid release were analyzed. The method showed high reproducibility (CVs 5.3%), good recovery rates (96–98%), and negligible in-vitro pre-analytical effects. The influence of in-vivo pre-analytical factors on TMAO levels was not observable within the applied experimental conditions. We did not find any correlation between TMAO levels and platelet activation at physiological TMAO concentrations, whereas platelet-derived eicosanoids presented activation of the cyclooxygenase and lipoxygenase pathways. In contrast to previously published results, we did not find any indications regarding diet dependency or circadian rhythmicity of TMAO levels. Our results do not support the hypothesis that TMAO increases platelet responsiveness via the release of lipid-mediators.

Dry etching is an important technique for pattern transferin fabrication of most opto-electronic devices, since it canprovide good control of both structure size and shape even on asub-micron scale. Unfortunately, this process step may causedamage to the material which is detrimental to deviceperformance. It is therefore an objective of this thesis todevelop and investigate low damage etching processes for InPbased devices. An ion beam system in combination with hydrocarbon (CH4) based chemistries is used for etching. At variousion energies and gas flows the etching is performed in twomodes, reactive ion beam etching (RIBE) and chemical assistedion beam etching (CAIBE). How these conditions affect both etchcharacteristics (e.g. etch rates and profiles, surfacemorphology and polymer formation) and etch induced damage (onoptical and electrical properties) is evaluated and discussed.Attention is also paid to the effects of typical post etchingtreatments such as annealing on the optical and electricalproperties. An important finding is the correlation betweenas-etched surface morphology and recovery/degradation inphotoluminescence upon annealing in PH3. Since this type of atmosphere is typical forcrystal regrowth (an important process step in III/Vprocessing) a positive result is imperative. A low ion energy N2/CH4/H2CAIBE process is developed which not onlysatisfies this criteria but also exhibits good etchcharacteristics. This process is used successfully in thefabrication of laser gratings. In addition to this, the abilityof the ion beam system to modify the surface morphology in acontrollable manner is exploited. By exposing such modifiedsurfaces to AsH3/PH3, a new way to vary size and density of InAs(P)islands formed on the InP surfaces by the As/P exchangereaction is presented. This thesis also proposes a new etch chemistry, namelytrimethylamine ((CH3)3N or TMA), which is a more efficient methyl sourcecompared to CH4because of the low energy required to break the H3C-N bond. Since methyl radicals are needed for theetching it is presumably a better etching chemistry. A similarinvestigation as for the CH4chemistry is performed, and it is found that bothin terms of etch characteristics and etch induced damage thisnew chemistry is superior. Extremely smooth morphologies, lowetch induced damage and an almost complete recovery uponannealing can be obtained with this process. Significantly,this is also so at relatively high ion energies which allowshigher etch rates. <b>Keywords:</b>InP, dry etching, ion beam etching, RIBE,CAIBE, hydrocarbon chemistry, trimethylamine, As/P exchangereaction, morpholoy, low damage, AFM, SCM, annealing

<p>Dry etching is an important technique for pattern transferin fabrication of most opto-electronic devices, since it canprovide good control of both structure size and shape even on asub-micron scale. Unfortunately, this process step may causedamage to the material which is detrimental to deviceperformance. It is therefore an objective of this thesis todevelop and investigate low damage etching processes for InPbased devices.</p><p>An ion beam system in combination with hydrocarbon (CH₄) based chemistries is used for etching. At variousion energies and gas flows the etching is performed in twomodes, reactive ion beam etching (RIBE) and chemical assistedion beam etching (CAIBE). How these conditions affect both etchcharacteristics (e.g. etch rates and profiles, surfacemorphology and polymer formation) and etch induced damage (onoptical and electrical properties) is evaluated and discussed.Attention is also paid to the effects of typical post etchingtreatments such as annealing on the optical and electricalproperties. An important finding is the correlation betweenas-etched surface morphology and recovery/degradation inphotoluminescence upon annealing in PH₃. Since this type of atmosphere is typical forcrystal regrowth (an important process step in III/Vprocessing) a positive result is imperative. A low ion energy N₂/CH₄/H₂CAIBE process is developed which not onlysatisfies this criteria but also exhibits good etchcharacteristics. This process is used successfully in thefabrication of laser gratings. In addition to this, the abilityof the ion beam system to modify the surface morphology in acontrollable manner is exploited. By exposing such modifiedsurfaces to AsH₃/PH₃, a new way to vary size and density of InAs(P)islands formed on the InP surfaces by the As/P exchangereaction is presented.</p><p>This thesis also proposes a new etch chemistry, namelytrimethylamine ((CH₃)₃N or TMA), which is a more efficient methyl sourcecompared to CH₄because of the low energy required to break the H₃C-N bond. Since methyl radicals are needed for theetching it is presumably a better etching chemistry. A similarinvestigation as for the CH₄chemistry is performed, and it is found that bothin terms of etch characteristics and etch induced damage thisnew chemistry is superior. Extremely smooth morphologies, lowetch induced damage and an almost complete recovery uponannealing can be obtained with this process. Significantly,this is also so at relatively high ion energies which allowshigher etch rates.</p><p><b>Keywords:</b>InP, dry etching, ion beam etching, RIBE,CAIBE, hydrocarbon chemistry, trimethylamine, As/P exchangereaction, morpholoy, low damage, AFM, SCM, annealing</p>