Conspicuous coloration is one of the main ways that animals communicate. The use of eye-catching color patterns to warn predators of an unprofitable trait is referred to as aposematism. Once predators learn to recognize the color pattern, a new signaling niche becomes available where other species can share the same signal. This mimicry niche can involve a “hide in plain sight” strategy by mimicking or parasitizing this signal, with mimics lacking the defense and associated costs that make them unprofitable. This is termed Batesian mimicry, and it decreases predation by taking advantage of the memory and learning of the predator community. Thus, a primary prediction in Batesian mimicry systems is that the model and mimic are found in sympatry. Another, fundamental prediction of Batesian mimicry is that the model outnumbers the mimic and that models emerge before the mimics to educate the predator guild. Some of these patterns were not significant in the California Coast Ranges as seen in Long et al., (2015), and no study has estimated population sizes for this temperate Batesian mimicry system. Furthermore, compared with community studies of mutualistic Müllerian mimicry in the tropics, no studies have tested predictions of parasitic Batesian mimicry on small scale patterns of habitat use and movement patterns. If mimicry is as an important part of the biology of these temperate species, as it is for their tropical counterparts, we predict that in addition to emerging first and being more abundant, the model and mimic will overlap strongly in habitat but the model will be more abundant in each habitat, and will move more and be more widespread among available habitats. Our results confirm these predictions and indicate that A. californica is effectively educating habitat specialist and generalist predators providing an umbrella of protection for the mimic L. lorquini.

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.

Increasingly, culturally and ethnically diverse environments provide an abundance of ordinary, everyday intergroup encounters, especially in public settings---often consisting of a conglomeration of positive and negative experiences. Yet few intergroup contact studies have focused on measuring and assessing contact in public settings. Reasons for this include both theoretical and methodological considerations. However, before the impact of mundane, everyday encounters on prejudice reduction can be assessed, it is necessary to examine the following questions: (1) Which situations are perceived as intergroup contact by participants? (2) How do individuals conceptualise where the boundaries for contact lie? (3) How are public and private forms of contact typically experienced? (4) Do they differ in the ways researchers have assumed in the past? and (5) Which methods are most appropriate for assessing public encounters? How can memory bias, temporality and locatedness be taken into account? Using qualitative, quantitative and near-time in-the-field methods, the following research examines these aspects in both public and private settings and provides first insights into how a novel method---the Contact Logger---can be used to assess the effects of public and private contact on attitudes. A three-day diary/interview study (N=17) explored how contact is experienced, understood and conceptualised in a variety of everyday intergroup contexts. This was followed by a survey study (N=525) that examined the boundaries of what is typically perceived as being contact. Insights from both studies fed into the development of a context-aware mobile application, which enabled the capturing of near-time intergroup encounters in situ. The usability of the resulting research tool---the Contact Logger---was tested, leading to further refinements. Following an initial feasibility study (N=104) that explored contact between young and older people, a field experiment (N=112) examined intergenerational contact in public and private contexts. Data collected with the Contact Logger were analysed on aggregate and day-to-day levels, and where possible compared to traditional retrospective survey data. Results from the first two studies indicated that while traditional intergroup encounters, such as contact with family and friends, are clearly conceptualised and viewed as contact, experiences and perceptions of contact in public settings are more disparate. Moreover, effects of such contact on attitudes are dependent on the idiosyncratic meaning attributed to the specific encounter as well as past experiences. Near-time data from a field-experiment (Study 5) provided evidence that intergroup encounters reported in situ compared to retrospective survey data differed in key variables (i.e., contact quality, duration, perceived status and group typicality). Correlational analyses between near-time and retrospective measures showed less correspondence than expected. Further, additional day-to-day analyses revealed that attitudes towards older people were less positive during weekends than weekdays, indicating that attitudes may be more dynamic than previously thought. Findings, as well as the different methodological and theoretical approaches, are critically discussed. Finally, a broad range of further applications for the Contact Logger are presented and important limitations are discussed.