The interplay between physiology and evolution in microorganisms is extremely relevant from the stand-point of human health, the environment, and biotechnology; yet microbial physiology and microbial evolution largely continue to grow as disjoint fields of research. The goal of this dissertation was to use experimental evolution to study methylamine metabolism in Methylobacterium extorquens species. Methylotrophs like the M. extorquens species grow on reduced single carbon compounds and are the largest biological sink for methane. M. extorquens AM1, the model system for the study of aerobic methylotrophy, has an unstable genome and severe growth defects as a result of laboratory domestication. First, I describe the genomic, genetic, and phenotypic characterization of a new model system for the study of aerobic methylotrophy: M. extorquens PA1. This strain has a stable genome, was recently isolated from a known ecological niche, and is closely related to AM1. Whereas PA1 grew 10-50% faster than AM1on most substrates, it was five-fold slower on methylamine. The PA1 genome encodes a poorly characterized but ecologically relevant N-methylglutamate pathway whereas AM1 also encodes the well-characterized methylamine dehydrogenase for methylamine oxidation. I characterized the genetics of the N-methylglutamate pathway in PA1 to resolve a linear topology that requires the formation of two, unique amino acid intermediates during methylamine oxidation. I also showed that methylamine metabolism via the N-methylglutamate pathway routes carbon flux in a manner completely different from previous instances of methylotrophy. Next, I evolved replicate populations of PA1 on methylamine for 150 generations. Based on the empirical heuristic that the initial fitness is negatively correlated to the rate of adaptation, it was expected that the fitness gain would be rapid. However, methylamine fitness did not improve at all; adaptive constraints led to evolutionary recalcitrance despite low initial fitness. These adaptive constraints were alleviated by the horizontal gene transfer of an alternate, functionally degenerate metabolic module. Finally, I uncovered ecologically distinct roles for two functionally degenerate routes for methylamine oxidation pathways in the AM1 genome; the highly expressed, efficient route is primarily used for growth and the tightly regulated, energetically expensive route is used for assimilating nitrogen in methylamine-limiting environments.

An intriguing mystery about tryptophan 2, 3-dioxygenase is its hydrogen peroxide-triggered enzyme reactivation from the resting ferric oxidation state to the catalytically active ferrous form. In this study, we found that such an odd Fe(III) reduction by an oxidant depends on the presence of L-Trp, which ultimately serves as the reductant for the enzyme. In the peroxide reaction with tryptophan 2, 3-dioxygenase, a previously unknown catalase-like activity was detected. A ferryl species (δ = 0.055 mm/s and ΔEQ = 1.755 mm/s) and a protein-based free radical (g = 2.0028 and 1.72 millitesla linewidth) were characterized by Mössbauer and EPR spectroscopy, respectively. This is the first compound ES-type of ferryl intermediate from a heme-based dioxygenase characterized by EPR and Mössbauer spectroscopy. Density functional theory calculations revealed the contribution of secondary ligand sphere to the spectroscopic properties of the ferryl species. A Trp-Trp dimer and a monooxygenated L-Trp were both observed as the enzyme reactivation by-products by mass spectrometry. Together, these results lead to the unraveling of an over 60-year old mystery of peroxide reactivation mechanism.

We conducted a computational adsorption study of methylamine on various surface-models of gold nanoparticle which is facetted by multiple {111} and {100} planes. In addition to these flat surfaces, our models include the stepped surfaces (ridges) formed along the intersections of these planes. Binding on the flat surface was fairly weak, but substantially stronger on the ridges by an average of 4.4 kcal/mol. This finding supports the idea that ssDNA’s interaction with gold nanoparticles occurs through the amines on the purine/pyrrimidine rings. Also, this typically undesirable interaction between DNA and gold nanoparticles is expected to increase as the particle size decreases. Our analysis suggests that particle size is an important controlling parameter to reduce this interaction. / Singapore-MIT Alliance (SMA)

La dégradation photocatalytique de deux amines, la méthylamine (MA) et la diméthylamine (DMA), a été étudiée en présence de TiO2 Degussa P25. Différents paramètres ont été étudiés: l'adsorption à l’obscurité et sous UV, la photolyse, les cinétiques de dégradation, l'effet du pH, l'effet de la nature et de l'intensité du flux photonique ainsi que les voies de dégradation de la MA et DMA.A l’obscurité, le taux de recouvrement des groupes OH est similaire pour la MA et DMA. Sous UV, ce taux devient deux fois plus élevé pour la MA. Les réactivités de MA et DMA sont directement corrélées à leur adsorption sous UV. Les atomes d'azote sont principalement décomposés en ammonium. Le nitrite a été également détecté, mais rapidement oxydé en nitrate. À pH basique, l'hydrolyse photo‐assisté et l'attaque de OH• sur l’atome N augmente. DMA est essentiellement transformé en MA. Les analyses du Carbone Organique Total (COT) montrent la présence de produits finaux contenant de l’azote difficilement minéralisables. Nous avons montré que, quelle que soit l'énergie des photons (UV‐A ou UV‐B), le rendement quantique reste constant et égal à 0,033.L'inactivation photocatalytique de E. coli en présence de la lumière solaire naturelle en absence (SODIS) et en présence de différents catalyseurs a été étudiée. L'effet de la température sur l'inactivation de E. coli a été aussi étudié. Les résultats ont montré que l’addition des différents types de catalyseurs accélère l'effet bactéricide du rayonnement solaire. Aucun phénomène de reviviscence bactérien n’a été observé après l'arrêt de l'exposition solaire durant au moins 72 heures. Seulement les ions ammonium et potassium ont été détectés au cours de l'inactivation de E. coli en accord avec la perforation de la membrane et l’oxydation des protéines. / The photocatalytic degradation of two amino‐compounds, methylamine (MA) and dimethylamine (DMA) was investigated in the presence of UV‐irradiated TiO2 aqueous suspensions. Different parameters were studied: adsorption under dark and UV conditions, photolysis, kinetics of degradation, effect of pH, effect of the nature and intensities of photonic flux and finally the chemical pathway MA and DMA degradation.While, the percentage of covered OH in the dark was equal for MA and DMA, it becomes twice higher for MA under UV. The reactivity of MA and DMA is directly correlated with the adsorption under UV. The nitrogen atoms were decomposed mainly to ammonium. Nitrite was also formed but was rapidly oxidized to nitrate. At basic pH, photo assisted hydrolysis and the attack of OH• on N‐atom increase. DMA is mainly transformed to MA. Total Organic Carbon (TOC) analysis show the presence of final slightly mineralised intermediate compounds containing nitrogen atom. We shown that, whatever the energy of photons (UV‐A or UV‐B), the same quantum yield equal to 0.033 was obtained.The photocatalytic inactivation of E. coli under natural solar irradiation in the absence (SODIS) as well in the presence of different concentrations of varied photocatalysts has also been investigated. The effect of temperature on E. coli inactivation was studied. Results show that the additions of any types of catalyst to the water accelerate the bactericidal action of solar irradiation and leads to a total disinfection. No bacterial regrowth was observed during the subsequent dark period. Ammonium and potassium ions were formed during E. coli inactivation in agreement with the membrane perforation and the oxidation of proteins.

A metilamina ('CH IND.3'NH IND.2') é um composto orgânico usado na produção de inseticidas, herbicidas, fungicidas, surfactantes, combustíveis fósseis, explosivos, produtos farmacêuticos, químicos fotográficos, tintas, tecidos, solventes, borrachas e anti-corrosivos. Estudos sobre tratamento de águas residuárias contendo metilamina são escassos e se restringem aos trabalhos envolvendo pesticidas carbamatados. Visando contribuir com os estudos acerca da degradação anaeróbia da metilamina, esta pesquisa estudou a comunidade microbiana e a atividade metanogênica específica em reatores anaeróbios em batelada, inoculados com lodo granular oriundo de reator UASB usado no tratamento de água residuária de abatedouro de aves, sob diferentes condições nutricionais: controle – sem metilamina, 5 mM, 10 mM, 20 mM, 30 mM, 50 mM, 75 mM e 90 mM de metilamina. Os reatores foram incubados sob temperatura de 30°C e agitação de 150 rpm. Desses reatores foram obtidas amostras para a determinação da atividade metanogênica específica (AME), sólidos suspensos voláteis (SVT), nitrogênio amoniacal e exames microscópicos. Ao final do experimento, foram realizados exames da biomassa por meio da técnica do número mais provável (NMP) e análise da diversidade microbiana por PCR/DGGE e seqüenciamento. O aumento da AME foi proporcional ao aumento das concentrações de metilamina, com inibição de produção de metano apenas nos reatores alimentados com 90 mM de metilamina. Os reatores alimentados com 50 mM e 75 mM de metilamina apresentaram os melhores resultados, com valores médios de AME de 0,0804 mmol 'CH IND.4'/g SVT.h e 0,0825 mmol 'CH IND.4'/g SVT.h respectivamente. Nos exames microscópicos foi verificado semelhança de morfologias microbianas em todas as concentrações de metilamina estudadas. Os organismos presentes nos reatores foram Methanosarcina sp., Methanosaeta sp., bacilos, coco-bacilos, filamentos e cocos. Em relação à análise de DGGE, não houve variação significativa nos padrões de bandas, tanto para o domínio Archaea quanto para o domínio Bacteria. Com os resultados da técnica de número mais provável (NMP) observou-se a predominância de arquéias metanogênicas dentre as bactérias anaeróbias totais. / The methylamine ('CH IND.3'NH IND.2') is an organic compound used in the production of insecticides, herbicides, fungicides, surfactants, fossil fuels, explosives, pharmaceuticals, photographic chemicals, paints, textiles, dyes, rubber and anticorrosive chemicals. Some studies about the treatment of wastewater containing methylamine are scarce and limited to works involving carbamate pesticides. This research aimed to study the anaerobic degradation of methylamine, the microbial community and the specific methanogenic activity in anaerobic battled reactors. The reactors were inoculated with granular sludge from a UASB reactor treating poultry wastes. Different nutritional conditions were adopted in the operation of the reactors: control (without methylamine), 5 mM, 10 mM, 20 mM, 30 mM, 50 mM, 75 mM and 90 mM of methylamine. The reactors were incubated under standard conditions: 30ºC and 150 rpm. Samples had been removed from the reactors to determine the specific methanogenic activity, the concentration of volatile suspended solids and ammoniacal nitrogen and the microscopic analysis. At the end of the experiment, the biomass was studied by the most probable number (MPN) technique and by the microbial diversity analysis with PCR and DGGE techniques. The increase of the specific methanogenic activity was proportional to the increase of methylamine concentration. The methane production was inhibited only in the reactor that was fed with 90 mM of methylamine. The reactors that were fed with 50 mM and 75 mM of methylamine showed the best results, with medium values of specific methanogenic activity equal to 0,0804 mmol 'CH IND.4'/g SVT.h and 0,0825 mmol 'CH IND.4'/g SVT.h, respectively. The microscopic analysis showed similarity between the microbial morphologies in all of the reactors. The observed microorganisms were Methanosarcina sp., Methanosaeta sp., rods, cocci and filaments. The DGGE analysis did not show significant variation in the standard profile of the Archaea and Bacteria domains. The results of the MPN technique revealed the predominance of the methanogenic archaea among the total anaerobic bacteria.

This thesis investigates the chemistry of ammonia borane (NH3BH3) relevant to the development of hydrogen storage systems for vehicular applications. Because of its high hydrogen content and low molecular weight ammonia borane has the potential to meet stringent gravimetric hydrogen storage targets of gt;9 wt%. Two of the three moles of H2 in ammonia borane can be released under relatively mild conditions, with the highest gravimetric yield obtained in the solid-state. However, ammonia borane does not deliver sufficient H2 at practical temperatures and the products formed upon H2 loss are not amenable to regeneration back to the parent compound. The literature synthesis of ammonia borane was modified to facilitate large scale synthesis, and the deuterated analogues ND3BH3 and NH3BD3 were prepared for the purpose of mechanistic studies. The effect of lithium amide on the kinetics of dehydrogenation of ammonia borane was assessed by means of solid-state reaction in a series of specific molar ratios. Upon mixing lithium amide and ammonia borane, an exothermic reaction ensued resulting in the formation of a weakly bound adduct with an H2N...BH3-NH3 environment. Thermal decomposition at or above temperatures of 50eg;C of this phase was shown to liberate gt;9 wt% H2. The mechanism of hydrogen evolution was investigated by means of reacting lithium amide and deuterated ammonia borane isotopologues, followed by analysis of the isotopic composition of evolved gaseous products by mass spectrometry. From these results, an intermolecular multi-step reaction mechanism was proposed, with the rates of the first stage strongly dependent on the concentration of lithium amide present. Compounds exhibiting a BN3 environment (identified by means of solid-state sup1;sup1;B NMR spectroscopy) were formed during the first stage, and subsequently cross link to form a non-volatile solid. Further heating of this non-volatile solid phase ultimately resulted in the formation of crystalline Li3BN2 - identified by means of powder X-ray diffractometry. This compound has been identified as a potential hydrogen storage material due to its lightweight and theoretically high hydrogen content. It may also be amenable to hydrogen re-absorption. The LiNH2/CH3NH2BH3 system was also investigated. Thermal decomposition occurred through the same mechanism described for the LiNH2/NH3BH3 system to theoretically evolve gt;8 wt% hydrogen. The gases evolved on thermal decomposition were predominantly H2 with traces of methane detected by mass spectrometry.

<p>Plasma activity of the enzyme semicarbazide-sensitive amine oxidase (SSAO; EC.1.4.3.6) has been reported to be high in disorders such as diabetes mellitus, chronic congestive heart failure and liver cirrhosis. Little is known of how the activity is regulated and, consequently, the cause for these findings is not well understood. Due to the early occurrence of increased enzyme activity in diabetes, in conjunction with the production of highly cytotoxic substances in SSAO-catalysed reactions, it has been speculated that there could be a causal relationship between high SSAO activity and vascular damage. Aminoacetone and methylamine are the best currently known endogenous substrates for human SSAO and the resulting aldehyde-products are methylglyoxal and formaldehyde, respectively. Both of these aldehydes have been shown to be implicated in the formation of advanced glycation end products (AGEs).</p><p>This thesis is based on studies exploring the regulation of SSAO activity and its possible involvement in the development of vascular damage. The results further strengthen the connection between high SSAO activity and the occurrence of vascular damage, since type 2 diabetic patients with retinopathy were found to have higher plasma activities of SSAO and lower urinary concentrations of methylamine than patients with uncomplicated diabetes. From studies on mice, it was also found that an SSAO inhibitor potently reduces the incorporation of methylamine-metabolite in the tissues. By quantifying SSAO-gene expression in alloxan-induced diabetes, increased transcription could be ruled out as a cause for the increased enzyme activity, thereby opening up for the possibility that the activity is regulated post-translationally. In fact, increased enzyme activity in adipose tissue was accompanied by decreased mRNA-levels, suggesting that the gene expression could be negatively controlled by the enzyme activity.</p>

Plasma activity of the enzyme semicarbazide-sensitive amine oxidase (SSAO; EC.1.4.3.6) has been reported to be high in disorders such as diabetes mellitus, chronic congestive heart failure and liver cirrhosis. Little is known of how the activity is regulated and, consequently, the cause for these findings is not well understood. Due to the early occurrence of increased enzyme activity in diabetes, in conjunction with the production of highly cytotoxic substances in SSAO-catalysed reactions, it has been speculated that there could be a causal relationship between high SSAO activity and vascular damage. Aminoacetone and methylamine are the best currently known endogenous substrates for human SSAO and the resulting aldehyde-products are methylglyoxal and formaldehyde, respectively. Both of these aldehydes have been shown to be implicated in the formation of advanced glycation end products (AGEs). This thesis is based on studies exploring the regulation of SSAO activity and its possible involvement in the development of vascular damage. The results further strengthen the connection between high SSAO activity and the occurrence of vascular damage, since type 2 diabetic patients with retinopathy were found to have higher plasma activities of SSAO and lower urinary concentrations of methylamine than patients with uncomplicated diabetes. From studies on mice, it was also found that an SSAO inhibitor potently reduces the incorporation of methylamine-metabolite in the tissues. By quantifying SSAO-gene expression in alloxan-induced diabetes, increased transcription could be ruled out as a cause for the increased enzyme activity, thereby opening up for the possibility that the activity is regulated post-translationally. In fact, increased enzyme activity in adipose tissue was accompanied by decreased mRNA-levels, suggesting that the gene expression could be negatively controlled by the enzyme activity.

A metilamina ('CH IND.3'NH IND.2') é um composto orgânico usado na produção de inseticidas, herbicidas, fungicidas, surfactantes, combustíveis fósseis, explosivos, produtos farmacêuticos, químicos fotográficos, tintas, tecidos, solventes, borrachas e anti-corrosivos. Estudos sobre tratamento de águas residuárias contendo metilamina são escassos e se restringem aos trabalhos envolvendo pesticidas carbamatados. Visando contribuir com os estudos acerca da degradação anaeróbia da metilamina, esta pesquisa estudou a comunidade microbiana e a atividade metanogênica específica em reatores anaeróbios em batelada, inoculados com lodo granular oriundo de reator UASB usado no tratamento de água residuária de abatedouro de aves, sob diferentes condições nutricionais: controle – sem metilamina, 5 mM, 10 mM, 20 mM, 30 mM, 50 mM, 75 mM e 90 mM de metilamina. Os reatores foram incubados sob temperatura de 30°C e agitação de 150 rpm. Desses reatores foram obtidas amostras para a determinação da atividade metanogênica específica (AME), sólidos suspensos voláteis (SVT), nitrogênio amoniacal e exames microscópicos. Ao final do experimento, foram realizados exames da biomassa por meio da técnica do número mais provável (NMP) e análise da diversidade microbiana por PCR/DGGE e seqüenciamento. O aumento da AME foi proporcional ao aumento das concentrações de metilamina, com inibição de produção de metano apenas nos reatores alimentados com 90 mM de metilamina. Os reatores alimentados com 50 mM e 75 mM de metilamina apresentaram os melhores resultados, com valores médios de AME de 0,0804 mmol 'CH IND.4'/g SVT.h e 0,0825 mmol 'CH IND.4'/g SVT.h respectivamente. Nos exames microscópicos foi verificado semelhança de morfologias microbianas em todas as concentrações de metilamina estudadas. Os organismos presentes nos reatores foram Methanosarcina sp., Methanosaeta sp., bacilos, coco-bacilos, filamentos e cocos. Em relação à análise de DGGE, não houve variação significativa nos padrões de bandas, tanto para o domínio Archaea quanto para o domínio Bacteria. Com os resultados da técnica de número mais provável (NMP) observou-se a predominância de arquéias metanogênicas dentre as bactérias anaeróbias totais. / The methylamine ('CH IND.3'NH IND.2') is an organic compound used in the production of insecticides, herbicides, fungicides, surfactants, fossil fuels, explosives, pharmaceuticals, photographic chemicals, paints, textiles, dyes, rubber and anticorrosive chemicals. Some studies about the treatment of wastewater containing methylamine are scarce and limited to works involving carbamate pesticides. This research aimed to study the anaerobic degradation of methylamine, the microbial community and the specific methanogenic activity in anaerobic battled reactors. The reactors were inoculated with granular sludge from a UASB reactor treating poultry wastes. Different nutritional conditions were adopted in the operation of the reactors: control (without methylamine), 5 mM, 10 mM, 20 mM, 30 mM, 50 mM, 75 mM and 90 mM of methylamine. The reactors were incubated under standard conditions: 30ºC and 150 rpm. Samples had been removed from the reactors to determine the specific methanogenic activity, the concentration of volatile suspended solids and ammoniacal nitrogen and the microscopic analysis. At the end of the experiment, the biomass was studied by the most probable number (MPN) technique and by the microbial diversity analysis with PCR and DGGE techniques. The increase of the specific methanogenic activity was proportional to the increase of methylamine concentration. The methane production was inhibited only in the reactor that was fed with 90 mM of methylamine. The reactors that were fed with 50 mM and 75 mM of methylamine showed the best results, with medium values of specific methanogenic activity equal to 0,0804 mmol 'CH IND.4'/g SVT.h and 0,0825 mmol 'CH IND.4'/g SVT.h, respectively. The microscopic analysis showed similarity between the microbial morphologies in all of the reactors. The observed microorganisms were Methanosarcina sp., Methanosaeta sp., rods, cocci and filaments. The DGGE analysis did not show significant variation in the standard profile of the Archaea and Bacteria domains. The results of the MPN technique revealed the predominance of the methanogenic archaea among the total anaerobic bacteria.

Ammonia excretion from the freshwater fish gill is thought to occur mainly via passive diffusion of NH3 aided by a favourable plasma-to-water ammonia gradient sustained by a pH gradient formed by an acidified gill boundary layer. Rhesus (Rh) proteins are the newest members of the ammonia transporter superfamily. In this thesis research, ten rainbow trout Rh cDNA sequences were cloned and characterized. Rhcg2 mRNA and H+-ATPase mRNA and activity levels were upregulated in the trout gill pavement cells in response to experimentally elevated plasma ammonia, concurrent with enhanced ammonia excretion. Controversially, Rh proteins are thought to transport C02. However, Rh mRNA levels in most tissues of hypercapnia-exposed trout remained stable suggesting that trout Rh proteins likely do not conduct C02. Xenopus oocytes expressing trout Rh proteins facilitated the bi-directional transport of methylamine, an ammonia analogue. Methylamine transport was inhibited by ammonia and sensitive to a pH gradient and the concentration of the protonated species. Use of the scanning ion electrode technique (SIET) indicated that trout Rh proteins have an ammonia affinity within the physiological range, which is greater than that for methylamine, and they transport ammonia more rapidly than methylamine. A model of ammonia excretion in the trout gill pavement cell is proposed wherein ammonia enters via basolateral Rhbg and exits via apical Rhcg2, binding to these channels as NH4+ but transiting as NH3. In the gill boundary layer, NH3 combines with an H+ ion released from H+-ATPase and/or Na+/H+ exchange, forming NH4+. As low-affinity, high-capacity ammonia transporters, Rh proteins in the trout gill would exploit the favourable pH gradient formed by the acidic boundary layer to facilitate rapid ammonia efflux when plasma ammonia levels are elevated. Basal plasma ammonia levels are likely maintained by simple passive NH3 diffusion with a smaller role for Rh proteins under these conditions. / Thesis / Doctor of Philosophy (PhD)