Development of a whole cell biochip for toxicant detection.

In the development of biosensors for ecotoxicity testing it is desirable to produce a small, portable system that can be used in the field. Toxicity testing using bioluminescence is widely used in the laboratory utilising natural and genetically modified (lux/ luc-marked) bacteria and other microorganisms. It is currently not possible to use genetically manipulated microorganisms in field testing and a biosensor, therefore, that incorporates naturally luminescent organisms may be preferred. In the development of a biosensor it is aimed to use the naturally luminescent bacterium Vibrio fischeri as a toxicity detection system on a chip. The bacterium will be immobilised in a polymeric matrix. Current work deals with the optimisation of light output and light preservation within the bacterium prior to immobilisation in polyvinyl alcohol. An examination of a range of physicochemical conditions within the polymer will be made, including cell density, thickness of polymer film, growth and light induction environment, and, preservation conditions, in order to develop a testing system giving consistent results over the lifetime of the biosensor. Data will be presented on light production using different culture media for the growth of V. fischeri and retention of light under immobilised conditions. .

Combustion Instability and Active Control: Alternative Fuels, Augmentors, and Modeling Heat Release

Experimental and analytical studies were conducted to explore thermo-acoustic coupling during the onset of combustion instability in various air-breathing combustor configurations. These include a laboratory-scale 200-kW dump combustor and a 100-kW augmentor featuring a v-gutter flame holder. They were used to simulate main combustion chambers and afterburners in aero engines, respectively. The three primary themes of this work includes: 1) modeling heat release fluctuations for stability analysis, 2) conducting active combustion control with alternative fuels, and 3) demonstrating practical active control for augmentor instability suppression. The phenomenon of combustion instabilities remains an unsolved problem in propulsion engines, mainly because of the difficulty in predicting the fluctuating component of heat release without extensive testing. A hybrid model was developed to describe both the temporal and spatial variations in dynamic heat release, using a separation of variables approach that requires only a limited amount of experimental data. The use of sinusoidal basis functions further reduced the amount of data required. When the mean heat release behavior is known, the only experimental data needed for detailed stability analysis is one instantaneous picture of heat release at the peak pressure phase. This model was successfully tested in the dump combustor experiments, reproducing the correct sign of the overall Rayleigh index as well as the remarkably accurate spatial distribution pattern of fluctuating heat release. Active combustion control was explored for fuel-flexible combustor operation using twelve different jet fuels including bio-synthetic and Fischer-Tropsch types. Analysis done using an actuated spray combustion model revealed that the combustion response times of these fuels were similar. Combined with experimental spray characterizations, this suggested that controller performance should remain effective with various alternative fuels. Active control experiments validated this analysis while demonstrating 50-70\% reduction in the peak spectral amplitude. A new model augmentor was built and tested for combustion dynamics using schlieren and chemiluminescence techniques. Novel active control techniques including pulsed air injection were implemented and the results were compared with the pulsed fuel injection approach. The pulsed injection of secondary air worked just as effectively for suppressing the augmentor instability, setting up the possibility of more efficient actuation strategy.

Non-invasive monitoring of changes in exhaled markers of airway inflammation in Thoroughbred racehorses

Exhaled breath (EB) and exhaled breath condensate (EBC) contain numerous volatile gases and a wide-array of non-volatile compounds, several of which have been investigated as markers of lower airway inflammation in human and veterinary medicine and have been used to diagnose and monitor diseases associated with pulmonary inflammation. The identification of reliable biomarkers within EB and EBC is an active research focus with the common goal of establishing non-invasive and repeatable assessment of respiratory health and disease in mammals. The application of EB and EBC analysis holds considerable appeal in the investigation of respiratory disease in Thoroughbred racehorses, as inflammatory airway disease (IAD) is a common cause for poor performance in this population of animals. This study documented that EB and EBC samples can be safely collected from Thoroughbred racehorses in their own environment, without adverse effect or interference with the horse’s training regimen. The use of off-line collection and analysis of exhaled gases via chemiluminescence is suitable for the measurement of exhaled carbon monoxide, but is not appropriate for analyzing exhaled nitric oxide in horses. Significant changes in the concentration of exhaled CO and the pH of EBC occurred in response to strenuous exercise and when exercising in different environmental temperatures. Exhaled CO was associated with tracheal mucus score (and the number of neutrophils in the mucus) and EBC pH was significantly different in horses with evidence of neutrophilic IAD compared to horses without IAD. Numerous physiological and environmental variables were identified as confounding factors in the assessment of both exhaled CO and EBC pH, with respiratory rate prior to EB collection, and during EBC collection, consistently identified as an explanatory variable influencing the concentration of exhaled biomarkers. Further studies in EB and EBC analysis in horses need to focus on objectively accounting for key respiratory dynamics during sample collection.

Salivary and serum cortisol levels, salivary alpha-amylase and unstimulated whole saliva flow rate in pregnant and non-pregnant

OBJETIVO: Comparar os níveis de cortisol sérico e salivar, alfa-amilase salivar (sAA) e fluxo de saliva não estimulada (UWS) em gestantes e não gestantes. MÉTODOS: Trata-se de um estudo longitudinal realizado no centro de promoção da saúde de um hospital universitário. Nove gestantes e 12 não gestantes participaram do estudo. Foram coletados e analisados soro e UWS nos três trimestres gestacionais e duas vezes por mês durante o ciclo menstrual. A análise do cortisol salivar e sérico foi realizada com o uso de quimiluminescência e a atividade da sAA foi determinada por meio de analisador automático para bioquímica. RESULTADOS: Foi verificado que a mediana (intervalo interquartil) dos níveis de cortisol sérico no grupo de gestantes foi maior que 23,8 µL/dL (19,4-29,4) quando comparado ao grupo de não gestantes, que teve média de 12,3 (9,6-16,8; p<0,001). Os níveis de sAA seguiram o mesmo padrão, com médias de 56,7 U/L (30,9-82,2) e 31,8 (18,1-53,2; p<0,001), respectivamente. Foram observadas diferenças dos níveis de cortisol sérico e salivar (µL/dL) e de sAA entre a fase folicular versus a fase lútea (p<0,001). As medianas dos fluxos salivares (UWS) foram semelhantes em gestantes (0,26 [0,15-0,30] mL/min) e não gestantes (0,23 [0,20-0,32] mL/min). Foram encontradas correlações significativas entre o cortisol salivar e o sérico (p=0,02) e entre o cortisol salivar e a sAA (p=0,01). CONCLUSÕES: Os níveis de cortisol sérico de sAA durante a gestação elevam-se. Na fase lútea do ciclo ovariano, os níveis de cortisol salivar aumentam ao passo que os níveis de cortisol sérico e sAA diminuem. _______________________________________________________________________________________ ABSTRACT

Anti-Helicobacter pylori activity and oxidative burst inhibition by the naphthoquinone 5-methoxy-3,4-dehydroxanthomegnin from Paepalanthus latipes

Helicobacter pylori is a bacterium recognized as the major cause of chronic gastritis and peptic ulcers. Infection by H. pylori induces inflammatory responses and pathological changes in the gastric microenvironment. The host Keywords: immune cells (especially neutrophils) release inflammatory mediators and large 5-methoxy-3,4-dehydroxanthomegnin amounts of reactive oxygen species (ROS), which are associated with an increased Helicobacter pyloririsk of developing gastric cancer. In this study, we evaluated the anti-H. pylori and oxidative burst antioxidantactivitiesofa1,4-naphthoquinone-5-methoxy-3,4-dehydroxanthomegnin. Paepalanthus latipes The antimicrobial activity was assessed using a spectrophotometric microdilution technique, and antioxidant activity was assessed by noting the effect of 5-methoxy3,4-dehydroxanthomegnin on the neutrophil oxidative burst using luminol-and lucigenin-amplified chemiluminescence. The results showed that 5-methoxy-3,4dehydroxanthomegnin is a potent anti-H. pylori compound (MIC 64 µg/mL and MBC 128 µg/mL) and a strong antioxidant. 5-Methoxy-3,4-dehydroxanthomegnin decreased luminol- and lucigenin-amplified chemiluminescence, with ED50 values of 1.58±0.09 µg/mL and 5.4±0.15 µg/mL, respectively, reflecting an inhibitory effect on the oxidative burst. These results indicate that 5-methoxy-3,4-dehydroxanthomegnin is a promising compound for the prevention and treatment of diseases caused by H. pylori infection, such as gastritis, peptic ulceration, and gastric cancer, because reactive oxygen intermediates are involved in the pathogenesis of gastric mucosal injury induced by H. pylori infections.

Leukocytes respiratory burst and lysozyme level in pacu (Piaractus mesopotamicus Holmberg, 1887)

Respostas imunológicas inatas são úteis para determinar o estado de saúde de peixes e avaliar o efeito de substâncias imunomoduladoras no cultivo destes animais. A atividade respiratória de leucócitos foi medida em pacu (Piaractus mesopotamicus) através de ensaio de quimioluminescência e ensaio de redução do nitroblue tetrazolium (NBT). O ensaio de redução do nitroblue tetrazolium pareceu mais adequado que o ensaio de quimioluminescência para determinação da atividade respiratória de leucócitos, uma vez que foi difícil isolar com êxito os leucócitos do sangue para o ensaio de quimioluminescência. Lisozima sérica e plasmática foram medidas por meio de ensaio turbidimétrico. Com o objetivo de inativar as proteínas do sistema complemento, as amostras de soro e plasma foram aquecidas (56 ºC por 30 minutos). Porém, este procedimento provocou a turvação das amostras de plasma e interferiu nos resultados. A atividade de lisozima no soro foi maior que no plasma, sugerindo que amostras de soro são mais apropriadas para esta análise. Este estudo estabeleceu protocolos que podem ser utilizados como ferramentas no estudo de mecanismos imunológicos do peixe tropical pacu.

Mixing, Chemical Reactions, and Combustion in Supersonic Flows

Experiments were conducted at the GALCIT supersonic shear-layer facility to investigate aspects of reacting transverse jets in supersonic crossflow using chemiluminescence and schlieren image-correlation velocimetry. In particular, experiments were designed to examine mixing-delay length dependencies on jet-fluid molar mass, jet diameter, and jet inclination.

The experimental results show that mixing-delay length depends on jet Reynolds number, when appropriately normalized, up to a jet Reynolds number of 500,000. Jet inclination increases the mixing-delay length, but causes less disturbance to the crossflow when compared to normal jet injection. This can be explained, in part, in terms of a control-volume analysis that relates jet inclination to flow conditions downstream of injection.

In the second part of this thesis, a combustion-modeling framework is proposed and developed that is tailored to large-eddy simulations of turbulent combustion in high-speed flows. Scaling arguments place supersonic hydrocarbon combustion in a regime of autoignition-dominated distributed reaction zones (DRZ). The proposed evolution-variable manifold (EVM) framework incorporates an ignition-delay data-driven induction model with a post-ignition manifold that uses a Lagrangian convected 'balloon' reactor model for chemistry tabulation. A large-eddy simulation incorporating the EVM framework captures several important reacting-flow features of a transverse hydrogen jet in heated-air crossflow experiment.

The photostability of wool doped with photocatalytic titanium dioxide nanoparticles

Photoyellowing of wool is a serious problem for the wool industry. This study assessed the role of photocatalytic nanocrystalline titanium dioxide (P-25) as a potential antagonist or catalyst in the photoyellowing of wool. Untreated, bleached and bleached and fluorescent-whitened wool slivers were processed into fine wool powders for the purpose of even and intimate mixing with the TiO2 nanoparticles in the solid state. Pure wool and wool/TiO2 mixtures were then compressed into solid discs for a photoyellowing study under simulated sunlight and under UVB and UVC radiations. Yellowness and photo-induced chemiluminescence (PICL) measurements showed that nanocrystalline TiO2 could effectively reduce the rate of photoyellowing by inhibiting free radical generation in doped wool, and that a higher concentration of TiO2 contributed to a lower rate of photooxidation and reduced photoyellowing. Hence nanocrystalline TiO2 acts primarily as a UV absorber on wool in dry conditions and not as a photocatalyst.

Elite hairy roots of Ocimum basilicum as a new source of rosmarinic acid and antioxidants

This study reports Agrobacterium rhizogenes-mediated transformation of three cultivars of Ocimum basilicum for hairy root establishment, screening and selection for the production of rosmarinic acid and antioxidants. Hairy root development was found to be explant-specific and virulence-dependent. Distinct inter-cultivar morphological variability was found between the seven axenically developed hairy root lines and morphological traits were found to be correlated with the presence of aux2 genes, their expression and endogenous IAA content. Further inter-cultivar variability in the content of total phenolics, rosmarinic acid and caffeic acid was also found. Production of rosmarinic acid was found to be age-dependent and cultivar-specific. Chemiluminescence analysis showed the hairy roots to be rich in antioxidants and that rosmarinic acid was the major antioxidant molecule. The concentration of rosmarinic acid was found to be positively correlated with the total antioxidant potential of the hairy root extracts. On the basis of origin, morphology and metabolite content, three elite hairy root lines were selected that had significantly higher rosmarinic acid production, biomass and antioxidant potential than non-transformed roots. These new lines are rich reserves of both antioxidants and rosmarinic acid.

Preservation of Nitric Oxide Availability as Nitrite and Nitrosothiols

Nitric Oxide (NO) has been known for long to regulate vessel tone. However, the close proximity of the site of NO production to “sinks” of NO such as hemoglobin (Hb) in blood suggest that blood will scavenge most of the NO produced. Therefore, it is unclear how NO is able to play its physiological roles. The current study deals with means by which this could be understood. Towards studying the role of nitrosothiols and nitrite in preserving NO availability, a study of the kinetics of glutathione (GSH) nitrosation by NO donors in aerated buffered solutions was undertaken first. Results suggest an increase in the rate of the corresponding nitrosothiol (GSNO) formation with an increase in GSH with a half-maximum constant EC50 that depends on NO concentration, thus indicating a significant contribution of ∙NO2 mediated nitrosation in the production of GSNO. Next, the ability of nitrite to be reduced to NO in the smooth muscle cells was evaluated. The NO formed was inhibited by sGC inhibitors and accelerated by activators and was independent of O2 concentration. Nitrite transport mechanisms and effects of exogenous nitrate on transport and reduction of nitrite were examined. The results showed that sGC can mediate nitrite reduction to NO and nitrite is transported across the smooth muscle cell membrane via anion channels, both of which can be attenuated by nitrate. Finally, a 2 – D axisymmetric diffusion model was constructed to test the accumulation of NO in the smooth muscle layer from reduction of nitrite. It was observed that at the end of the simulation period with physiological concentrations of nitrite in the smooth muscle cells (SMC), a low sustained NO generated from nitrite reduction could maintain significant sGC activity and might affect vessel tone. The major nitrosating mechanism in the circulation at reduced O2 levels was found to be anaerobic and a Cu+ dependent GSNO reduction activity was found to deliver minor amounts of NO from physiological GSNO levels in the tissue.

Physicochemical complexity in complex chemical systems

Self-replication and compartmentalization are two central properties thought to be essential for minimal life, and understanding how such processes interact in the emergence of complex reaction networks is crucial to exploring the development of complexity in chemistry and biology. Autocatalysis can emerge from multiple different mechanisms such as formation of an initiator, template self-replication and physical autocatalysis (where micelles formed from the reaction product solubilize the reactants, leading to higher local concentrations and therefore higher rates). Amphiphiles are also used in artificial life studies to create protocell models such as micelles, vesicles and oil-in-water droplets, and can increase reaction rates by encapsulation of reactants. So far, no template self-replicator exists which is capable of compartmentalization, or transferring this molecular scale phenomenon to micro or macro-scale assemblies. Here a system is demonstrated where an amphiphilic imine catalyses its own formation by joining a non-polar alkyl tail group with a polar carboxylic acid head group to form a template, which was shown to form reverse micelles by Dynamic Light Scattering (DLS). The kinetics of this system were investigated by 1H NMR spectroscopy, showing clearly that a template self-replication mechanism operates, though there was no evidence that the reverse micelles participated in physical autocatalysis. Active oil droplets, composed from a mixture of insoluble organic compounds in an aqueous sub-phase, can undergo processes such as division, self-propulsion and chemotaxis, and are studied as models for minimal cells, or protocells. Although in most cases the Marangoni effect is responsible for the forces on the droplet, the behaviour of the droplet depends heavily on the exact composition. Though theoretical models are able to calculate the forces on a droplet, to model a mixture of oils on an aqueous surface where compounds from the oil phase are dissolving and diffusing through the aqueous phase is beyond current computational capability. The behaviour of a droplet in an aqueous phase can only be discovered through experiment, though it is determined by the droplet's composition. By using an evolutionary algorithm and a liquid handling robot to conduct droplet experiments and decide which compositions to test next, entirely autonomously, the composition of the droplet becomes a chemical genome capable of evolution. The selection is carried out according to a fitness function, which ranks the formulation based on how well it conforms to the chosen fitness criteria (e.g. movement or division). Over successive generations, significant increases in fitness are achieved, and this increase is higher with more components (i.e. greater complexity). Other chemical processes such as chemiluminescence and gelation were investigated in active oil droplets, demonstrating the possibility of controlling chemical reactions by selective droplet fusion. Potential future applications for this might include combinatorial chemistry, or additional fitness goals for the genetic algorithm. Combining the self-replication and the droplet protocells research, it was demonstrated that the presence of the amphiphilic replicator lowers the interfacial tension between droplets of a reaction mixture in organic solution and the alkaline aqueous phase, causing them to divide. Periodic sampling by a liquid handling robot revealed that the extent of droplet fission increased as the reaction progressed, producing more individual protocells with increased self-replication. This demonstrates coupling of the molecular scale phenomenon of template self-replication to a macroscale physicochemical effect.