We hypothesized that the exercise-induced increase in muscle tricarboxylic acid (TCA) cycle intermediates (TCAI) would be lower after aerobic training (TR), due to a better match between pyruvate production and subsequent oxidation and lower flux through the alanine aminotransferase (AAT) reaction. Eight men [22 ± 1 y; maximal aerobic capacity (V02max) = 3.9 ± 0.2 L/min] cycled at 75% of their pre-TR V02max to exhaustion (Exh), before and after 7 wk ofTR (1 hr/d, 5 d/wk). Muscle biopsies (v. lateralis) were obtained at rest, 5 min of exercise and Exh. The effect ofTR was evidenced by an increased time to fatigue (91 ± 6 vs 42 ± 6 min), increases in resting [glycogen] and citrate synthase maximal activity, and decreases in glycogen degradation, lactate accumulation and phosphocreatine utilization during exercise. The sum of 4 measured TCAI was similar between trials at rest, but lower after 5 min of exercise post- TR (2. 7 ± 0.2 vs 4.3 ± 0.2 mmol.kg-1 dw, P<0.05). Importantly, the [TCAI] at Exh post- TR (2.9 ± 0.2 mmol.kg-1 dw) was not different compared to 5 min of exercise and thus fatigue was not attributable to a decline in TCAI. The net change in glutamate (Post: 4.5 ± 0.7 vs Pre: 7.7 ± 0.6 mmol.kg-1 dw) and alanine (Post: 3.3 ± 0.2 vs Pre: 5.6 ± 0.3 mmol.kg-1 dw) from Rest-5 min of exercise was attenuated post-TR (P<0.05), which is consistent with lower flux through the AA T reaction. We conclude that changes in muscle TCAI during exercise are not causally related to aerobic energy provision. / Thesis / Master of Science (MSc)

The dynamical behaviors of tricarboxylic acid derivative, 1,3,5-tris(carbox- ymethoxy) benzene[TCMB, C6H3(OCH2COOH)3] on an Au surface is investigated by molecular dynamics. A TCMB molecule adsorbed on the Au(111) substrate is first probed into the structure arrangement. It founds that there are four possible conformations of the TCMB molecule that is adsorbed on the Au(111) substrate. The main difference on its conformation is the orientation of its functional group, which lead the molecule that forms the lock-and-key (LAK) behavior and prompts the molecule that become more stable on the substrate. As this result, the LAK behavior directly affects the trajectories of movement and dynamical behaviors. Another topic is to observe the behavior of TCMB molecule on Au(110) and Au(100) surface, respectively. As well as the result of the TCMB molecule adsorbed on the Au(111) substrate, it also shows a different behavior on dynamical behaviors when the TCMB molecule adsorbed on the Au(110) and Au(100) substrate. Moreover, we found that the diffusion direction of TCMB molecule is dependent on the arrangement of the adsorbed surface. From the observation of the trajectory of the TCMB molecule, we found that diffusion range is most wide on Au(100) plane. The translational direction of TCMB molecule tend to move on the <110> direction as the molecule is migrate on the Au(100) plane, whereas that tend to move on the [1ī0] direction as the molecule is migrate on the Au(110) plane. From the description above, we know that TCMB molecule with different conformations on different plane of surface arrangement displays different trajectories of movement and dynamical behaviors. Therefore, in order to understand the dynamical behaviors of TCMB monolayer on gold surface. In this work, the temperature effect on the adsorption behavior and the dynamic behavior of TCMB monolayer structure on the Au(111) substrate are investigated. From the calculation of the cohesive energy between molecules and the interaction energy between the molecule and the Au(111) substrate, we found that there are significant changes in cohesive energy and interaction energy at specific temperatures, which can be attributed to the deformation of the monolayer structure. Finally, the mean square displacement (MSD), diffusion coefficient(D) and distance between the molecule and the Au(111) substrate are calculated to investigate the diffusion property and motion behavior of TCMB monolayer at specific temperatures.

No description available.

This dissertation, studies two specific topics related to the research of surface science by employing the molecular dynamics (MD) approach, that of a water droplet deposited on a poly (methyl methacrylate) (PMMA) substrate and that of a single tricarboxylic acid derivative, 1, 3, 5-tri(carboxymethoxy) benzene (TCMB, C6H3(OCH2COOH)3 ) adsorbed on gold (100) and (110) surfaces. These can help engineers clarify the characteristics and phenomena of physical adsorption of the molecule, as well as contributing to the application of surface science. This work is divided into two parts. Effect of droplet size on the structural and dynamical behavior of a water droplet spreading on a PMMA amorphous surface: note that most experts prefer to consider a rigid model as the substrate in research of surface wetting because it is more efficient to run the MD simulation such that a long simulation can be accomplished in a short time. The results verify that the rigid model is not suitable to act as the PMMA substrate in simulation because it prevents the diffusion of PMMA molecules, which then affects the penetration behavior of water molecules in the droplet upon impact with the PMMA surface. Several sizes of water droplets are considered in order to understand the size influence of the droplet on the properties of water molecules and on the PMMA surface. The penetrated water molecules and the local roughness increase with a decrease in the size of the droplet, which also leads to a smaller contact angle of the water droplet on the PMMA substrate. When the droplet is composed of more than 1000 water molecules, the contact angle shows agreement with experimental results. As regarding the structure of the water molecule in the droplet on PMMA substrate, the average number of hydrogen-bonded penetrating water molecules is in inverse proportion to the size of the droplet By examining the velocity field, the regular motion of the water droplet is found during the equilibrium process and after the droplet reaches the equilibrium state. The diffusion of the water molecules shows a significant decrease for the penetrated water molecules and an increase as it gradually approaches the vapor/liquid interface. Finally, calculations at different regions are made for the vibration spectrum of the oxygen atom, life time, and the relaxation time of the hydrogen bond. The changes of the hydrogen-bond dynamics of the hydrogen bond are consistent with the change of the distribution of the hydrogen bond angle. Effect of surface structure on the structural and dynamical behavior of a tricarboxylic acid derivative molecule on Au surfaces: the dynamical behavior of the single tricarboxylic acid derivative, 1, 3, 5-tri(carboxymethoxy) benzene (TCMB, C6H3(OCH2COOH)3 ) on Au (100) and (110) surfaces by molecular dynamics simulation approach is studied to provide better understanding of surface diffusion. Four possible conformations of the adsorbed TCMB molecule on the Au surface are found, with differences arising from different numbers of CH2 groups adsorbed on the Au substrate. Both the number of CH2 groups in the TCMB molecule that interact with Au surface and the different geometric relationship between the TCMB molecule and the Au surface strongly affect the translational motion, rotational motion, interaction energy and the Lock-and-Key behaviors of the TCMB molecule. A poor complementarity between the TCMB molecule shape and atomic structure of the surface results in significant migration of the molecule and is therefore an unstable adsorption. These results will be useful for the design of a molecular monolayer.

Short-chain fatty acids (SCFAs) and the tricarboxylic acid (TCA) cycle metabolites aresmall hydrophilic compounds that play crucial roles in biological species ranging fromenergy metabolism, immune homeostasis to cellular signalling. There is a need for reliableand precise quantification of these metabolites in biological matrices as they can providecrucial information of metabolic status and potentially be used as diagnostic biomarkersfor different pathological and physiological conditions. However, their retention andseparation in traditional reversed-phase system, without chemical derivatization, is oftenproblematic due to their volatile and hydrophilic characteristics. The aim of this studywas to implement a facile and effective derivatization method for the simultaneousquantitation of SCFAs and TCA cycle metabolites by LC-qToF-MS in negative ion mode. Inthis work, 3-nitrophenylhydrazine (3-NPH) was employed for preanalyticalderivatization to convert the compounds to their respective 3-nitrophenylhydrazones.Analytical standards and faecal samples were used to assess the linearity, matrix effect,accuracy, extraction efficiency, precision, retention-time shift and short-term stability.The compounds were successfully separated within 6 minutes on a reverse-phase C18column. All the compounds showed good linearity (R2≥ 0.97) in both solvent-only andfaecal samples. The matrix effect was minimal and did not affect the compoundsquantitation. The extraction efficiency ranged from 80% to 110% (CV≤9.7%, n = 6). Theaccuracy of quantitation was determined to be between 82.8% to 113.8% (CV≤9.0%, n =6). The intra-day (CV%) demonstrated good precision for all analytes, the inter-day (%)were more variable due to the derivatives’ chemical instability. However, most of thederivatives were chemical stable up to 5 days in the autosampler (10°C). The method wasalso applied to explore the levels of these metabolites in human faecal samples and mousebrain samples.

The gut microbiota plays a major part in maintaining the health of a human host. Countless of crucial functions in the body, including immune responses, cell signaling and energy metabolism to name a few, are conducted by the gut microbiota and its metabolites. Accordingly, it is of interest to gain knowledge on what can alter the gut microbiota, as these alterations by extension can give rise to adverse health effects. In this study, the impact of polychlorinated biphenyl (PCB)- and organochlorine pesticide (OCP) exposure on tricarboxylic acid (TCA) cycle metabolites, short-chain fatty acids (SCFAs) and bile acids, as well as other polar and semi-polar metabolites, which are all related to the gut microbiota, were investigated. An in vitro fermentation of faecal samples exposed to a PCB/OCP mixture was performed, and liquid chromatography-time of flight mass spectrometry (LC-qToF-MS) targeted and non-targeted approaches were applied to the extracts. The results obtained suggested that PCBs and OCPs most likely have an effect on the levels of several features of the gut metabolome with either increased or decreased levels upon exposure. Bile acids and TCA metabolites appear to follow a trend of decreasing levels, while no apparent effects could be seen for the SCFAs. Furthermore, distinct concentrations of the PCB/OCP mixture appear to induce different changes in gut microbiota functioning, which highlights the importance of performing dose-response studies when exploring biological effects of these compounds. The identification of different metabolite profiles during fermentation also allows for the possibility of further investigation of potential biomarkers to assess PCB/OCP exposure.

Endophytic microorganisms are important sources of bioactive compounds and have been widely studied due to the properties that their metabolites have. Compared to the host plants are easier handling in the laboratory. From the Phomopsis sp fungus genus have been isolated compounds with different biological activities. The investigation of the metabolites, whether primary or secondary, present in the filtrate of culture is of utmost importance. The metabolomic study of Phomopsis sp can lead to understanding of the production pathways of the metabolites of interest. The objective of the present work was to trace the metabolic profile of the filtrate from the endophytic fungus Phomopsis sp., An isolate of Syzygium jambolanum DC, through the consumption of dextrose as a source of carbon and energy for 6 weeks of culture using the 1H NMR With water signal suppression by NOESY 1D. In addition, liquid column chromatography and thin layer chromatography were performed in order to isolate compounds from organic mycelium extract. The analyzes had the help of MatLab® software for data normalization. Thus, it was possible to trace the metabolic profile of Phomopsis sp., cultivated under conditions of hypoxia, and verify the occurrence of reductive routes such as alcoholic fermentation and reductive Tricarboxylic Acid Cycle. It was also possible to observe the intensification of yellow pigmentation, characteristic of this genus of fungus, from the fourth week of cultivation, which is the period most of the primary metabolites are no longer present. By means of NMR experiments it was possible to elucidate a structure of a substance isolated from the chloroform extract of the fungus dry mycelium, 5-methylmethane. Based on these results, it can be inferred that, under these conditions, the microorganism studied performs alcoholic fermentation throughout the culture. It was also verified the occurrence of the reductive Tricarboxylic Acid Cycle in the cytosol until the third week of culture and, after this period, from the fourth week the secondary metabolism is intensified. It is the first time that the secondary metabolite 5-methylmeleina is isolated from this genus of endophytic. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Microrganismos endofíticos, que são importantes fontes de compostos bioativos, vêm sendo amplamente estudados devido às propriedades que seus metabólitos apresentam e, em comparação com as plantas hospedeiras, são de mais fácil manipulação em laboratório. Visto que do gênero de fungo endofítico Phomopsis já foram isolados compostos com diferentes atividades biológicas, a investigação dos metabólitos, sejam eles primários ou secundários, presentes no filtrado de sua cultura é de extrema importância. O estudo metabolômico de Phomopsis sp. pode levar ao entendimento das vias de produção dos metabólitos de interesse. O presente trabalho teve como objetivo traçar o perfil metabólico do filtrado da cultura do fungo endofítico Phomopsis sp., um isolado de Syzygium jambolanum DC, através do consumo da dextrose como fonte de carbono e energia durante 6 semanas de cultivo utilizando a técnica de RMN 1H com supressão do sinal da água por NOESY 1D. O enfoque do estudo é. Além disso, foram realizadas cromatografia líquida em coluna e cromatografia em camada delgada com o intuito de isolar compostos a partir de extrato orgânico do micélio. As análises estatísticas tiveram o auxílio dos softwares MatLab® para normalização e quantificação relativa dos dados. Assim, foi possível traçar o perfil metabólico de Phomopsis sp, cultivado em condições de hipóxia, e constatar a ocorrência de rotas redutivas, tais como fermentação alcoólica e Ciclo dos Ácidos Tricarboxílicos redutivo. Também foi possível observar a intensificação da pigmentação amarela, característica desse gênero de fungo, a partir da quarta semana de cultivo, período este que a maioria dos metabólitos primários já não se fazem mais presentes. Através de experimentos de RMN foi possível elucidar a estrutura de uma substância isolada do extrato em clorofórmio do micélio seco do fungo, a 5-metilmeleina. Com base nesses resultados, pode-se inferir que, sob estas condições, o microrganismo estudado realiza fermentação alcoólica durante todo o cultivo. Também foi verificada a ocorrência do Ciclo dos Ácidos Tricarboxílicos redutivo no citosol até a terceira semana de cultivo e, após esse período, a partir da quarta semana o metabolismo secundário é intensificado. É a primeira vez que o metabólito secundário 5-metilmeleina é isolado desse gênero de endofítico.

No description available.

Pseudomonas aeruginosa is a Gram-negative, metabolically versatile, opportunistic pathogen that exhibits a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability is in part mediated by two-component systems (TCS) that play a crucial role in regulating virulence mechanisms, metabolism and antibiotic resistance. Our sequence analysis of the P. aeruginosa PAO1 genome revealed the presence of two open reading frames, mifS and mifR, which encodes putative TCS proteins, a histidine sensor kinase MifS and a response regulator MifR, respectively. This two-gene operon was found immediately upstream of the poxAB operon, where poxB encodes a chromosomal ß-lactamase, hinting at the role of MifSR TCS in regulating antibiotic resistance. However, loss of mifSR had no effect on the antibiotic resistance profile when compared to P. aeruginosa parent PAO1 strain. Subsequently, our phenotypic microarray data (BioLOG) and growth profile studies indicated the inability of mifSR mutants to grow in α-ketoglutarate (α-KG), a key tricarboxylic acid (TCA) cycle intermediate, as a sole carbon source. To date, very little is known about the physiology of P. aeruginosa when provided with α-KG as its sole carbon source and the role of MifS and MifR TCS in virulence. Importantly, in the recent years, α-KG has gained notoriety for its newly identified role as a signaling molecule in addition to its conventional role in metabolism. This led us to hypothesize that MifSR TCS is involved in α-KG utilization and virulence in P. aeruginosa. Using mifS, mifR and mifSR clean in-frame deletion strains, our study demonstrates that the MifSR TCS modulates the expression P. aeruginosa kgtP (PA5530) and pcaT (PA0229) genes encoding putative α-KG permeases. In addition, our study shows that the MifSR-regulation of these transporters requires functional sigma factor RpoN (σ54). Loss of mifSR in the presence of α-KG, resulted in differential regulation of P. aeruginosa key virulence determinants including biofilm formation, motility, cell cytoxicity and the production of pyocyanin and pyoverdine. Involvement of multiple regulators and transporters suggests the presence of an intricate circuitry in the transport of α-KG and its importance in P. aeruginosa survival. This is further supported by the α-KG-dependent MifSR regulation of multiple virulence mechanisms. Simultaneous regulation of multiple mechanisms involved in P. aeruginosa pathogenesis suggests a complex mechanism of MifSR action. Understanding the physiological cues and regulation would provide a better stratagem to fight often indomitable P. aeruginosa infections.