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Most in-service Ni-based turbine disc alloys have a bi-modal or tri-modal γʹ size distribution, where primary γʹ size (or presence) is linked to the duration and temperature of the solution heat treatment (e.g. super or sub-solvus) whilst secondary γʹ and tertiary γʹ size are linked to the ageing temperature and the cooling rates. The effect and relative balance of these different γʹ size populations on slip character is a matter of continuing debate. Therefore it is necessary to establish the role of γʹ size on slip character for unimodal distributions to allow a deeper understanding of deformation behaviour. The accumulation of damage is intrinsically affected by the degree of slip heterogeneity at both low and high temperatures and this can affect both crack initiation and growth processes. In this work, a non-commercial heat treatment has been used on RR1000 alloy to produce two different unimodal γʹ size distributions. The heat treatments also caused a varying carbide distribution on grain boundary, with more continuous grain boundary carbides observed in the coarse γʹ variant. These carbides on grain boundaries also play a key role in strengthening and weakening damage mechanisms over a range of temperatures. Short fatigue crack tests (uninterrupted and interrupted tests) have been conducted on polished plain bend bars of both fine and coarse γʹ variants under three point bending, with a sine waveform (20Hz) and trapezoidal waveform (90s dwell time) loading applied at room temperature and 650° in air, respectively. At room temperature, in terms of deformation and slip character, it is seen that the fine γʹ variant exhibits more γʹ shearing and heterogeneous slip character (relatively higher planar slip and intense slip bands), which is more linked to less resistance to crack initiation while the coarse γʹ variant is apt to show more Orowan looping and homogeneous slip character. In both γʹ variants fatigue cracks mainly initiated at pores at the surface or sub-surface, slip bands or along twin boundary presenting a crystallographic faceting. High strain levels and more diffuse strain localisation take place in the vicinity of crack initiation areas such as pore and slip bands. Crack initiation along the twin boundary occurs with relatively high Schmid factor (SF) and further propagation is inclined to follow high SFs of adjacent grains with the activated slip systems. Short fatigue crack growth rates in both γʹ variants show a high extent of scatter at low ΔK level as a distinctive short crack behaviour, which is significantly linked to the complexity of local microstructure and intrinsic shielding effects. Under the testing conditions in this study, the lifetimes appear to be strongly connected to the number of crack initiation sites (partly linked to initiation resistance), which seems more associated with the defect distribution or grain misorientation than γʹ alone. More crack initiation sites generally lead to the multi-site coalescence events that contribute to enhanced fatigue crack growth rates and reduced lifetime. Three dimensional (3D) evaluation (sub-surface) of early crack growth processes by X-ray CT scanning has also been conducted on micro-tensile samples of the coarse γʹ variant at room temperature. Internal microstructure has been investigated from a series of tomographically reconstructed two dimensional (2D) slices assessing porosity distribution and complex morphology of the early crack, showing the crack propagated along slip bands slanted at around 45°. Overall, the lifetimes at 650°C are significantly shortened by the effect of dwell time and consequent oxidation damage. At elevated temperature, fatigue crack initiation mechanisms are influenced by synergistic effects of the microstructural features and oxidation processes. Cross slip and homogenous slip character are promoted in both γʹ variants due to the effects of elevated temperature. On applying a 90s dwell time, a large amount of grain boundary cracking at bulging Co-rich oxides can be seen on the surface in both γʹ variants. Such plentiful intergranular cracking promotes frequent crack coalescences and results in significantly accelerated fatigue crack growth rates, leading to shortened lifetime. The bulged oxides at particular grain boundaries are associated with enhanced mismatch strain and stress concentration, resulting in oxide cracking. Cracks also initiate at the interface between oxidised carbides and grain boundaries due to volume expansion and concomitant strain localisation. The complex influences of microstructural variation, loading frequency (linked to diffusion time), temperature and oxidation on long fatigue crack growth behaviour have also been assessed. Long fatigue crack tests on single edge notched bend samples of both γʹ variants have been carried out under three point bend and a trapezoidal waveform loading with dwell times of 1s and 90s at peak load at 650°C and 725°C in air. Overall, in both γʹ variants, intergranular failure modes are dominant and even more marked with longer dwell time and higher temperature. The fine γʹ variant shows a better fatigue resistance at 650°C and 725°C for both dwell times, and this is more marked with longer dwell time and higher temperature. The coarse γʹ variant exhibits more time dependent crack growth and distinctly more intergranular fracture modes, which can ascribed to the more continuous carbide distribution on grain boundaries. Oxidation damage zones ahead of the crack tip in both γʹ variants were investigated by switching between low and high loading frequencies at 650°C, showing that the coarse γʹ variant exhibited the most significant oxidation damage zone in the transition. Such damage zones are linked to embrittlement cracking and accelerated crack growth rates. 3D analysis by X-ray CT scanning as well as 2D observation showed that the crack path at low frequency (longer diffusion time per cycle) is relatively more tortuous and contains greater secondary crack formation, with a discontinuous crack path and uncracked ligaments seen due to significant oxidation damage and embrittled zones formed which is more marked in the coarse γʹ variant.

In bacterial transcription, transcription initiation is arguably the most important regulatory point, because transcribing unnecessary genes into RNA could be a waste of energy, time and resources. There are multiple components which are involved in bacterial transcription initiation: RNA polymerase, [sigma]-factors, transcription factors, and transcription start sites. Each component has been intensively investigated, however in a limited scope and mostly with low-throughput methods. New technologies, such as hybridization on microarray and deep-sequencing, enabled researchers to study each component in a systems level, in a combination of two or more components, and in comparison between different species. In order to facilitate the analysis, integration, and comparison, software, MetaScope, was developed to accommodate multiple genome-scale datasets to visualize, analyze, integrate, and compare. TSS-seq, modified 5'-RACE with deep- sequencing, gave a genome-scale landscape of transcription start sites, and comparison of TSSs of conserved genes between closely-related species, E. coli and K. pneumoniae, showed significantly different usage of promoters, which implies different regulation of orthologous genes. To further investigate properties of promoters which were identified by TSS-seq, ChIP-chip experiments were performed for [sigma]-factors in E. coli to determine [sigma]-factor regulons. From the reconstructed [sigma]- factor network, extensive overlaps between regulons were observed. [sigma]⁷⁰ and [sigma]³⁸ share the largest set of genes in E. coli, and additional experiments revealed that those [sigma]-factors work in competition and utilize the negative regulation by [sigma]³⁸ . ChIP-exo, which applies exonuclease to present better resolution of DNA-binding, and RNA-seq implemented more detailed identification of Fur regulon in E. coli. Reconstruction of Fur regulon completed the previous knowledge of bacterial response to iron change, and also enabled its role over iron metabolism. In order to understand how bacteria respond to nitrogen limitation, the same methods were used under conditions that were predicted from model-based prediction, and resulted in reconstruction of regulons for major transcription factors, NtrC and Nac. Determination of those regulons expanded the current knowledge of nitrogen metabolism and how it is regulated in bacteria. Thus, systems approaches enabled a genome-scale assessment of regulatory components in multiple levels, and contributed to expansion of the current knowledge of bacterial transcription initiation

The human immune system is a complex network of molecules, organelles, cells, tissues, and organs - there are an uncountable number of interactions and transformations interconnecting all the system components. In addition to such biochemical components, pressure, flow, morphology and location of all of such biophysical components also play an important role in the human immune system. While technical difficulties have frequently limited consideration of the immune system component interactions, some state-of-the-art analytical techniques have revealed distinct cellular behaviors that occur only in vivo or in vivo-mimetic environments. These types of findings inspire analytical/bioanalytical chemists to provide new tools to mimic such interactions occurring in the human body and facilitate better understanding of the human immune system. Use of microfluidics in analytical/bioanalytical science is a relatively recent development but provides significant potential for such purpose. Well-designed microfluidic approaches can reveal both biophysical and biochemical aspects of human in vivo systems with quick and temporally, spatially, and chemically resolved analyses of in vitro assays. The aim of this thesis is to enable the study of cellular behaviors in physiologically relevant environments using microfluidics. Chapter 1 of this thesis provides a brief review of microfluidics-based innovations in cellular biology studies, and the following chapters focus on biophysical (e.g. flow, Chapter 2), biochemical (e.g. multiple chemical signals, Chapter 3), and higher level biological networks (e.g. cell-cell interaction, Chapter 4) and describe how microfluidics-based approaches advance our current understanding of cellular behaviors. Furthermore, this thesis suggests a potential future direction of microfluidics-based assay platforms by incorporating a powerful detection scheme, surface-enhanced Raman scattering (SERS), into a microfluidic platform (Chapter 5). Fluid flow is ubiquitous in the human body, and thus, the first part of the thesis focuses on the flow-induced biophysical impact on cellular behaviors. The biological model herein is the human blood vessel, and the cytotoxicity of nanoparticles under varying flow conditions is investigated in the context of its impact on cellular viability and adhesion/aggregation behaviors. Mesoporous silica nanoparticles are used as the model nanoparticles and human endothelial cells and platelets are used as the model cell types to mimic human blood vessels. Compared to traditional cytotoxicity assays performed under static conditions, mesoporous silica nanoparticles show higher and shear stress-dependent toxicity to endothelial cells under flow conditions. Furthermore, nanoparticles slightly compromise platelet adhesion to endothelial cells at low nanoparticle doses; however, high nanoparticle doses significantly increase the number of platelet adhesion events, leading to higher probability for uncontrolled platelet actions (e.g. clot formation in vivo). Interestingly, despite the altered level of platelet adhesion and aggregation, in no case did nanoparticle exposure result in significant loss of platelet viability. This work clearly demonstrates that 1) biophysical aspects such as fluid flow have to be considered in studying cellular behaviors and 2) aspects besides viability, such as cellular adhesion and interaction with other cell types, have to be considered in the context of nanotoxicology. The simple and highly adaptable microfluidic analytical platform used in this chapter will be useful for further studies involving other nanoparticles, different cell types, and different cell functions. Besides biophysical aspects such as flow, cells in the human body always interact with numerous chemical signals and other cell types around them - this thesis also investigates these interactions. By using a model of chemotaxis, chemically directed cellular migration, the dynamics of cellular behaviors under multiple chemical signals (Chapter 3) and in the context of cell-cell interactions (Chapter 4) were quantitatively studied using a microfluidic platform. The target cells used in these chapters are neutrophils, the most abundant and motile leukocyte in humans. The microfluidic platform establishes a stable and dynamic gradient of chemicals across a cell culture chamber and enables the quantitative investigation of cellular migration in the presence of multiple chemical signals. This study leads to interesting findings such as a hierarchy of neutrophil response to chemical signals, the involvement of an enzyme in regulating neutrophils to decide which signal to follow, and the involvement of endothelial cells in regulating neutrophil chemotaxis through surface marker expression. In addition, this study also reveals the impact of neutrophil activation in neutrophil chemotaxis, providing insights into the potential of neutrophil chemotaxis regulation for therapeutic purposes. This study brings new knowledge about neutrophil chemotaxis by replicating physiologically relevant environments for neutrophils (e.g. multiple chemical signals in the context of cell-to-cell communications), and yields both fundamental and therapeutically relevant insight. In parallel to above mentioned biochemical studies with in vivo mimetic microfluidic platforms, this thesis also aims to achieve microfluidics-based analytical platforms with powerful chemical identification capability. Microfluidics facilitates parallel, low volume sample manipulation and provides opportunities for various optical signal transduction mechanisms. Chapter 5 of this thesis provides proof-of-concept microfluidic sensor platforms that fully utilize such advantages of microfluidics. The developed microfluidic sensor platforms are equipped with gold-based SERS substrates that enable highly sensitive chemical fingerprinting, and the microfluidic device enables serial dilution of introduced analyte solution that terminates in five discrete sensing elements. Chapter 5 demonstrates the utility of the developed sensor platforms to create a calibration curve or do a limit of detection study in a single experiment. Overall, the thesis herein advances our current understanding of cellular behaviors occurring inside the human body, and demonstrates the utility and a potential future direction of microfluidics-based experimental platforms. All findings described in this thesis will facilitate better understanding of the human immune system and lead to new development of medical and pharmacological applications.