The core of the dissertation lies in developing two novel systems bioengineering approaches, a synthetic Escherichia coli killer-prey microecology, and a combined infection-inflammation NET-array system, to investigate the role of the mechanochemical complexity of the microenvironment in driving the microbe-microbe and host-microbe interactions, respectively. Herein, the first part of the dissertation includes designing and engineering a synthetic E. coli killer-prey microecological system where we quantified the quorum-sensing mediated interactions between the engineered killer and prey E. coli bacterial strains plated on nutrient-rich media. In this work, we developed the plate assay followed by plasmid sequencing and computational modeling that emphasizes the concept of the constant evolution of species or acquired resistance in the prey E. coli, in the vicinity of the killer strain. We designed the microecological system such that the killer cells (dotted at the center of the plate) constitutively produce and secrete AHL quorum-sensing molecules into the microenvironment. AHL then diffuses into the prey cells (spread throughout the plate) and upregulates the expression of a protein that lyses the prey. Through time-lapse imaging on petri plates automated using a scanner, we recorded the "kill wave" that originates outside the killer colony and travels outward as the prey dies. We found that the prey population density surrounding the killer decreased in comparison to other locations on the plate far from the killer. However, some of the prey colonies evolve to be resistant to the effects of AHL secreted by the killer. These prey colonies resistant to the killer were then selected and confirmed by plasmid sequencing. Using this empirical data, we developed the first ecological model emphasizing the concept of the constant evolution of species, where the survival of the prey species is dependent on the location (distance from the killer) or the evolution of resistance. The importance of this work lies in the context of the evolution of antibiotic-resistant bacterial strains and in understanding the communication between the microbial consortia, such as in the gut microbiome. Further, the second part of the dissertation includes quantifying the interactions between immune cells (primary healthy human neutrophils) and motile Pseudomonas aeruginosa bacteria in an inflammation-rich microenvironment. Neutrophils, being the first responding immune cells to infection, defend by deploying various defense mechanisms either by phagocytosing and killing the pathogen intracellularly or through a suicidal mechanism of releasing their DNA to the extracellular space in the form of Neutrophil Extracellular Traps (NETs) to trap the invading pathogens. Although the release of NETs is originally considered a protective mechanism, it is shown to increase the inflammation levels in the host if unchecked, ultimately resulting in end-organ damage (especially lung and kidney damage), as with the severe cases of sepsis and COVID-19. In our work, we developed a combined infection-inflammation NET-array system integrated with a live imaging assay to quantify the spatiotemporal dynamics of NET release in response to P. aeruginosa infection in an inflammatory milieu at a single-cell resolution. Importantly, we found increased NET release to P. aeruginosa PAO1 when challenged with inflammatory mediators tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), but not leukotriene B4 (LTB4), compared to the infection alone. Our device platform is unique in that the nanoliter well-assisted individual neutrophil trapping enables us to quantify NET release with single-cell precision. Besides, incorporating confined side loops in the device helped us study the role of mechanical confinement on NET release, showing reduced NET release from neutrophils confined in the side loops compared to the relatively wider chambers of our microsystem. In summary, our work emphasizes the importance of studying the heterogeneity of NET release in host defense and inflammation. In the future, our system can be used for screening novel neutrophil-based immunotherapies and serve as a valuable research tool in precision medicine. / Doctor of Philosophy / The microenvironment plays a vital role in shaping the interactions within microbes and between the host and the microbes. Microbes use quorum-sensing-based chemical signaling to adapt to the environmental stresses in a microecology (be it a soil microecology or the gut microbiome). They communicate with each other with the help of these chemicals to regulate their population density (to mutual benefit in the case of a biofilm formation or to compete for resources in the case of a predator-prey model). In the first part of the dissertation, we utilize this quorum-sensing approach to study the spatiotemporal dynamics of the interactions between two engineered killer and prey Escherichia coli bacterial strains on a nutrient-rich agar plate in real-time. We designed the microecological system such that the killer cells (dotted at the center of the plate) constitutively produce and secrete AHL quorum-sensing molecules into the microenvironment. AHL then diffuses into the prey cells (spread throughout the plate) and upregulates the expression of a protein that lyses the prey. We found that the prey population density surrounding the killer decreased in comparison to other locations on the plate far from the killer. Further, through sequencing, we found that some of the prey colonies acquired resistance to the effects of AHL secreted by the killer. We then developed a computational model that recapitulates our experimental results, emphasizing the concept of the constant evolution of species or acquired resistance. The importance of this work lies in using experimental and computational approaches to better understand the evolution of multidrug-resistant (MDR) bacterial strains. Next, we investigated the interactions between primary human neutrophils (first responding immune cell type to infection) and motile Pseudomonas aeruginosa bacteria in the second part of the dissertation, explicitly focusing on quantifying neutrophil extracellular traps (NETs) release. With increasing concerns regarding the role of the dysregulated NET release in exaggerated inflammatory responses in the host, it is imperative to quantify NET release precisely at a single-cell level in a controlled microenvironment. To this end, we engineered a combined infection-inflammation NET-array device with 1024 nanoliter wells per device and achieved single-cell level trapping of neutrophils in these wells. Our device platform is unique in that the individual wells of the device have constricted side loops, which helps us better understand the role of mechanical confinement on NET release from an engineering standpoint. We then used the NET-array system to quantify the spatiotemporal dynamics of NET release to P. aeruginosa in an inflammatory mediator-rich microenvironment. Importantly, we found heightened NET release to Pseudomonas aeruginosa PAO1 when challenged with inflammatory mediators tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), but not leukotriene B4 (LTB4), compared to the infection alone. We also demonstrated reduced NET release from neutrophils confined in the side loops compared to the relatively wider chambers of our combined infection-inflammation microsystem. Especially with the increasing complexity of the intercellular cues at the site of infection, by integrating our microfluidic method with the conventional reductionist approaches, we can better solve the intricate puzzles of the immune cell decision-making processes at a single-cell level. Our study highlights the importance of fine-tuning NET release in controlling pathological neutrophil-driven inflammation.

Sediment is one of the leading non-point source pollutants in the U.S and has detrimental effects on biological communities such as aquatic communities; human use such as recreation; and natural processes such as flood water storage. For silvicultural operations, the majority of sediment is produced from erosion on highly disturbed areas, such as skid trails, haul roads, and log landings. Erosion from silvicultural activities not only has the potential to introduce sediment into waterways but can also decrease site productivity through the removal of topsoil. In order to minimize erosion from silvicultural operations, forestry Best Management Practices (BMPs) have been developed, but efficacies of various BMP options are not well documented. This study evaluated five closure and cover BMPs for the control of erosion on bladed skid trails through both field based measurements with sediment traps and soil erosion modeling. The erosion models used were the Universal Soil Loss Equation for Forestry (USLE – Forest), the Revised Universal Soil Loss Equation version 2 (RUSLE2), and the Water Erosion Prediction Project for Forest Roads (WEPP – Forest Roads). Erosion model predictions were also regressed against field based results to determine accuracy. The bladed skid trail BMP treatments evaluated were: 1) water bar only (Control); 2) water bar and grass seed (Seed); 3) water bar, grass seed, and straw mulch (Mulch); 4) water bar and piled hardwood slash (Hardwood Slash); and 5) water bar and piled pine slash (Pine Slash). Field based results show that the Control treatment was the most erosive (137.7 tonnes/ha/yr), followed by the Seed treatment (31.5 tonnes/ha/yr), Hardwood Slash treatment (8.9 tonnes/ha/yr), Pine Slash treatment (5.9 tonnes/ha/yr), and finally the Mulch treatment was the most effective erosion control technique (3.0 tonnes/ha/yr). Model accuracy results show that RUSLE2 performed the best overall. Both USLE – Forest and WEPP – Forest Roads under predicted values on the Control treatment, where erosion rates were very high. WEPP – Forest Roads under predicted these values the most. All models generally show that the Control was the most erosive followed by the Seed, Hardwood Slash, Pine Slash, and Mulch treatments. / Master of Science

One of the greatest challenges in finfish aquaculture is combating losses caused by infectious bacterial diseases, and a better understanding of the interactions between the host immune system and pathogens is essential for developing new methods to manage infections and outbreaks. Extracellular traps (ETs) are decondensed nuclear chromatin released by neutrophils into the extracellular matrix that can ensnare and kill microbes. Since the discovery of ETs in humans, these innate immune effectors have been characterised across the animal kingdom, including in some fish species, though their existence the salmonids has yet to be confirmed. Therefore, the aim of this thesis was to confirm and characterise the release of ETs in the rainbow trout (Oncorhynchus mykiss) and investigate the interaction of these structures with fish pathogenic bacteria. To do this, a triple-layer Percoll gradient technique was employed to give highly enriched cell suspensions of polymorphonuclear cells (PMNs) derived from head-kidney tissue preparations. Treatment of PMN-enriched cell suspensions with the nucleic-acid-specific stain, SYTOX Green, revealed the presence of ET-like structures that had been released without stimulation. These ET-like structures were confirmed by immunostaining techniques to contain the diagnostic proteinaceous markers of ETs: neutrophil elastase, myeloperoxidase and the H2A histone. Previously characterised inhibitors and inducers of ET release from phagocytic immune cells in other animals confirmed that calcium ionophore (CaI), flagellin, and cytochalasin D shared similar activities for ET-release by rainbow trout PMNs. However, interestingly, as the common ET-inducer phorbol-myristate acetate (PMA) and ET-inhibitor diphenyleneiodonium (DPI) did not exert their expected potency in ET release assays with the PMNs, perhaps indicating that these fish cells are less dependent on NADPH oxidase signalling for ET release compared to mammals and most invertebrate species. The PMN-derived ETs were demonstrated to bind to and trap the extracellular nuclease-deficient bacterial fish pathogen, Vibrio anguillarum (Vib 87) when co-cultured. Finally, extracellular nuclease activity produced by a V. anguillarum isolate (Vib 6) during culture was able to degrade ETs released by rainbow trout PMNs in a dose-dependent manner. Moreover, viable colony counts, fluorescent and phase contrast microscopy demonstrated that V. anguillarum Vib 6 eluded trapping by ETs, while an extracellular nuclease-deficient isolate did not. These observations are consistent with the suggestion that nucleases are a microbial virulence factor during host infection. Confirming the existence and antimicrobial potential of extracellular traps released by rainbow trout PMNs may provide a platform towards the development of novel therapeutics to reduce mortalities in finfish aquaculture caused by infectious microbial pathogens.

A new depth-based method of seismic imaging is used to provide insights into the 3D structural geometry of faults, and to facilitate a detailed structural interpretation of the Penola Trough, Otway Basin, South Australia. The structural interpretation is used to assess fault kinematics through geological time and to evaluate across-fault juxtaposition, shale gouge and fault reactivation potential for three selected traps (Zema, Pyrus and Ladbroke Grove) thus providing a full and systematic assessment of fault seal risk for the area. Paper 1 demonstrates how a depth-conversion method was applied to two-way time seismic data in order to redisplay the seismic in a form more closely representative of true depth, here termed ‘pseudo-depth’. Some apparently listric faults in two-way time are demonstrated to be planar and easily distinguishable from genuine listric faults on pseudo-depth sections. The insights into fault geometry provided by pseudo-depth sections have had a significant impact on the new structural interpretation of the area. Paper 2 presents the new 3D structural interpretation of the area. The geometry of faulting is complex and reflects variable stress regimes throughout structural development and the strong influence of pre-existing basement fabrics. Some basement-rooted faults show evidence of continual reactivation throughout their structural history up to very recent times. Structural analysis of all the live and breached traps of the area demonstrate that traps associated with a basement rooted bounding fault host breached or partially breached accumulations, whereas non-basement rooted faults are associated with live hydrocarbon columns. Papers 3 and 4 demonstrate that for all the traps analysed (Zema, Pyrus and Ladbroke Grove), initial in-place seal integrity was good. The initial seal integrity was provided by a combination of both favourable across fault juxtaposition (Ladbroke Grove) and/or sufficiently well developed shale gouge over potential leaky sand on sand juxtaposition windows to retain significant hydrocarbon columns (Zema, Pyrus). The palaeocolumns observed at Zema and Pyrus indicate that there has been subsequent post-charge breach of seal integrity of these traps while Ladbroke Grove retains a live hydrocarbon column. Evidence of open, permeable fracture networks within the Zema Fault Zone suggest that it is likely to have recently reactivated, thus breaching the original hydrocarbon column. Analysis of the in-situ stress tensor and fault geometry demonstrates that most of the bounding faults to the selected traps are at or near optimal orientations for reactivation in the in-situ stress tensor. The main exception being the Ladbroke Grove Fault which has a NW-SE trending segment (associated with a relatively high risk of fault reactivation and possible leakage at the surface) and an E-W trending segment (associated with a relatively low risk of fault reactivation and a present day live column). The free water level of the Ladbroke Grove accumulation coincides with this change in fault orientation. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339545 / Thesis (Ph.D.) - University of Adelaide, Australian School of Petroleum, 2008

This thesis presents axially symmetric equivalents of three-dimensional rf ion traps. Miniaturization in mass spectrometry has focused on miniaturizing mass analyzers. Decrease in mass analyzer size facilitates reduction of the size of other components of a mass spectrometer, especially the radio frequency electronics and vacuum system. Miniaturized mass analyzers are made using advanced microfabrication techniques. Due to micromachining limitations, it is not possible to fabricate ion traps with exact axial symmetry. The motivation for this thesis is to investigate newer three-dimensional geometries which do not possess axial symmetry, but are equivalent in performance to axially symmetric ion traps. We introduce a 3D geometry called square ion trap(SIT) having a ring electrode made off our square shaped planar surfaces and square shaped endcap electrodes resembling a cuboid. Initially, a SIT geometry is taken and it will be investigated if this unknown 3D geometry can be made equivalent to a well characterized, axially symmetric ion trap like the CIT. The purpose of showing equivalence will be to understand the ion dynamics and fields inside the new 3D SIT. This thesis consists of five chapters. In Chapter 1, we present the necessary background information required to understand the operation of a mass spectrometer. The Paul trap geometry is introduced followed by the derivation of equation of ion motion inside the Paul trap. The Mathieu stability plot and the modes of operation of a mass spectrometer are briefly discussed. The chapter ends by outlining scope of the thesis. Chapter 2 describes the computational methods employed by us in the thesis. First, the geometry of square ion trap is introduced. Then the boundary element method(BEM) which is used to compute the charge distribution on the electrode surfaces is discussed. This is followed by the three-dimensional Green’s function which should be employed for non-axially symmetric structures. The method to calculate potential and field inside the ion trap from charge distribution is shown. Calculation of multipole coefficients for non-axially symmetric traps using charge distribution is shown. The methods used to generate ion trajectory and stability plot are discussed. The Nelder-Mead simplex method used for optimization is also presented. To verify our numerical methods of charge calculation, we have taken standard textbook problems and compared our results with those presented therein. The multipoles calculation, field and ion trajectory was verified by comparing the results for the Paul trap and cylindrical ion traps. Chapter 3 presents the results for axially symmetric equivalents of 3D rf ion traps. SIT geometry of dimensions equivalent to the CIT0 are taken and field and multipoles are studied in it. Then optimization is applied to create a CIT geometry equivalent to the SIT under study. Axial field and ion trajectory was compared and observed to be matching. Finally, stability plot was generated for both SIT and its equivalent CIT and was found to present a close match. Chapter 4 presents the numerical results obtained for three-dimensional rf ion trap equivalent of CIT. In this chapter, we have considered two standard geometries, the CIT0 and the CITopt. Optimization was applied to create SIT geometries equivalent to the CIT0 and the CITopt respectively. Comparison of fields and ion trajectory confirmed the fact that non-axially symmetric traps can be created equivalent to any axially symmetric ion trap. We have also considered another case of axially symmetric circular planar ion trap which has an annular ring electrode and two planar endcap electrodes. Square equivalent of circular planar trap was created by the optimizer and its equivalent was verified by ion trajectory comparison. Chapter 5 summarizes the thesis with a few concluding remarks.

Globally, as many as 30% of amphibians and 23% of reptile species are threatened and require urgent conservation action. The major threats are primarily caused by anthropogenic activities. This research aimed to investigate the impacts of two anthropogenic threats, over-exploitation and habitat changes on herpetofaunal populations in Hong Kong. Platysternon megacephalum has been heavily depleted because of severe hunting pressure for the food and pet trades. Low densities of individuals and few remaining intact populations have limited our ability to study their ecology. I conducted mark-recapture and radio-telemetry studies on P.megacephalum populations in five streams in Hong Kong, one of which was free from trapping, over 34 months (2009–2011) to investigate the impacts of illegal trapping on populations of P.megacephalum and their spatial ecology and growth. Illegal trapping was associated with the absence of large adults, smaller body sizes of adults and skewed ratios of juveniles to adults. Home ranges were relatively small with a mean 100% minimum convex polygon 996m2. Males moved longer distances than females and both sexes moved longer distances in wet seasons. P.megacephalum was highly aquatic, preferring to stay in pools, and their microhabitat preferences were affected by stream width and depth, and substrate types. Juvenile P.megacephalum grew rapidly, with growth declining after attainment of sexual maturity. The average ages of sexual maturation were eight years for females. Illegal trapping remains the major threat to P.megacephalum populations and I recommend that regulatory personnel identify key streams and patrol regularly against illegal trapping to safeguard remaining populations. In Hong Kong, large areas of primary forest have been replaced by secondary forests and plantations, and changes in tree species composition have often led to alteration of associated plant and animal communities. I examined the herpetofaunal assemblages in secondary forests and exotic Lophostemon confertus plantations. Amphibian abundances were higher in secondary forests while reptile abundance, species richness of amphibian and reptiles were similar. Secondary forests provided better habitats for amphibians and I recommend the planting of a mixture of native tree species and the thinning of exotic trees in future plantation management efforts in South China. Our knowledge about the effectiveness of different herpetofaunal survey methods in Southeast Asia is limited. To fill the information gap, I examined the effectiveness of three survey methods, including transect surveys, pitfall traps and coverboards, for sampling terrestrial herpetofauna. Transect surveys were most effective at sampling species richness and pitfall traps were most efficient in capturing high numbers of reptiles. The results of this study will aid researchers in assessing the feasibility of and choosing herpetofaunal survey methods in Southeast Asia. Despite the severe threats that herpetofauna are facing, our understanding of their ecology and conservation needs remains limited. More research and the initiation of monitoring programs for herpetofauna, strengthened enforcement of existing regulations, and proper habitat management are crucial for the conservation of herpetofauna in South China. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

No description available.

Organic electronics has evolved into a well-established research field thanks to major progresses in material sciences during recent decades. More attention was paid to this research field when “the discovery and development of conductive polymers” was awarded the Nobel Prize in Chemistry in 2000. Electronic devices that rely on tailor-made material functionalities, the ability of solution processing and low-cost manufacturing on flexible substrates by traditional printing techniques are among the key features in organic electronics. The common theme while exploring organic electronics, and the focus of this thesis, is that (semi-)conducting polymers serve as active materials to define the principle of operation in devices. This thesis reviews two kinds of organic electronic devices. The first part describes electrochemical devices based on conducting polymers. Active matrix addressed displays that are printed on flexible substrates have been obtained by arranging electrochemical smart pixels, based on the combination of electrochemical transistors and electrochromic display cells, into cross-point matrices. The resulting polymer-based active-matrix displays are operated at low voltages and the same active material is used in the electrochemical transistors as well as in the electrochromic display cells, simply by employing the opto-electronic properties of the material. In addition to this first part, a switchable optical polarizer based on electrochromism in a stretch-aligned conducting polymer is described. The second part reports switchable charge traps in polymer diodes. Here, a device based on a solid-state blend of a conjugated polymer and a photochromic molecule has been demonstrated. The solid state blend, sandwiched between two electrodes, provide a polymer diode that allows reversible current modulation between two different charge transport mechanisms via externally triggered switching of the charge trap density.

O nordeste paulista é composto por um mosaico de ambientes fortemente antropizado, com predominância de plantios de cana-de-açúcar, além de florestas plantadas de eucalipto e fragmentos de Cerrado. Embora o veado-catingueiro (Mazama gouazoubira) seja uma espécie amplamente distribuída em diferentes biomas do Brasil, pouco é conhecido sobre a sua capacidade de adaptação em paisagens alteradas. Neste contexto, modelos de ocupação da paisagem e dados de armadilhas fotográficas foram utilizados para verificar quais fatores ambientais são importantes na escolha do uso e seleção de hábitat pelo veado-catingueiro. Foram amostradas quatro áreas em três cenas de paisagens, entre os meses de Abril e Setembro de 2013 e 2014, totalizando um esforço amostral de 6240 armadilhas/dia. Os resultados apontam que o veado-catingueiro está amplamente distribuído nos remanescentes de Cerrado do nordeste do estado de São Paulo, além de ocupar com a mesma intensidade tanto o interior como o entorno de unidades de conservação. Foi observado um efeito positivo dos plantios de silvicultura na ocupação da paisagem pela espécie. A distribuição temporal dos registros ao longo do ciclo de 24 horas também foi semelhante dentro e fora de unidades de conservação. Estes resultados sugerem que o veado-catingueiro é uma espécie ecologicamente plástica e que está se beneficiando da heterogeneidade ambiental presente no nordeste paulista. Estes hábitos generalistas lhe atribuem vantagens em comparação a outras espécies mais especialistas nesse cenário. / The northeast of São Paulo state is a region where Cerrado remants are immersed in a landscape with high human influence mediated by vast sugarcane and Eucalyptus plantations. Mammals survival in this landscape has been little explored. Brown brocket deer (Mazama gouazoubira, Fischer 1814) is a species with widespread distribution in different biomes of Brazil, however little is known about its capacity of adaptation in landscapes with intensive agriculture. We conducted a study based on occupancy modelling from camera trapping data in order to evaluate which environmental factors are important to habitat selection by brown brocket deer. The study was carried out in three agricultural landscapes at four study areas between April and September (2013 and 2014) with a total effort of 6240 traps-day. Our results show that brown brocket deer are well adapted to Cerrado/agricultural matrix in the state of Sao Paulo, showing high ecological flexibility. This deer used non-protected areas as much as protected areas. Silviculture represented the only landscape covariate with high relative importance for brown brocket deer. Also, brown brocket deer shows rhythm of activity similar in and out protected areas. Our results suggest that brown brocket deer is extremely plastic species that is benefiting from environmental heterogeneity present in São Paulo\'s northeast. The generalist habit of this deer gives it advantage when compared to other more specialist species in this scenario.

Na periodontite, um potencial loop inflamatório oral-sistêmico é identificado pela presença de produtos da atividade neutrofílica em locais onde o processo inflamatório não foi desenvolvido inicialmente. Nossos resultados mostram um aumento da quantidade de neutrófilos circulantes, assim como a resposta destas células dos animais com periodontite induzida por ligadura (7 dias) ante diferentes estímulos in vitro em comparação aos animais Sham. A produção de ânion superóxido por neutrófilos circulantes de ratos com periodontite foi maior quando estimulados com PMA ou Zimosan. Na liberação de MPO, PMA, Zimosan ou fMLP produziram efeitos maiores no grupo com periodontite. A formação de NETs foi maior nos neutrófilos dos ratos com periodontite em resposta ao PMA. Além das respostas estimuladas, a liberação de MPO e a formação de NETs em condições basais foram maiores nos neutrófilos provenientes dos ratos com periodontite. Estes resultados mostram que existe uma pre-ativação (priming) dos neutrófilos circulantes já na fase inicial do desenvolvimento da periodontite. / In periontitis, a potential loop of oral-systemic inflammation is identified by the presence of neutrophil activity products at the sites where the inflammatory process was not initially developed. Our results show an increase in the amount of circulating neutrophils, as well as the response of these cells from rats with ligature-induced periodontitis (7 dias) to different in vitro stimuli, in comparison with those obtined from Sham animals. Superoxide anion production by circulating neutrophils from rats with peridontitis was higher when stimulated with either PMA or Zymosan. In MPO release, either PMA, zymosa or fMLP produced greater effects by periodontitis group. The formation of NETs was also higher in the neutrophils from rats with peridontitis in response to PMA. In addition to the stimulated responses, under basal conditions, MPO release and NET formation were also higher in neutrophils obtained from rats with periodontitis. These results show that circulating neutrophils are pre-activated (primed) even in the initial phase of periodontitis development.