Despite the highly acidic environment of the human stomach, Helicobacter pylori inhabits the gastric mucosa of half of the world population. While a fraction of the infected individuals develop gastric diseases, the presence of H. pylori has also been linked to benefits such as protection against allergies. The pathogenic and beneficial aspects of the bacteria require intricate balance in its motility, colonization and interaction with the host. In this dissertation I focus on understanding the factors that influence motility. The first part of this dissertation presents a comparison of the microrheology and the bacterial motility in healthy and tumor human gastric mucins. Active bacteria motion led to shear-thinning of the mucin solution and decreased its viscosity. The tumor mucin showed the lowest viscosity and its microrheology was most affected by bacterial motion. The second part of the dissertation elucidates the interplay between acidity and gastric mucin microrheology on motility. H. pylori motility shows a non-monotonic pH dependence, with the median of speed distribution peaking at pH4 in aqueous broth, below which the flagella motors fail due to high external proton concentration and bacteria become immotile below pH3. In contrast, in mucin the viscosity dominates motility; the median speed peaks at pH5 related to the approaching sol-gel transition around pH4. Additionally, the cell rotation increases monotonically with decreasing pH in mucin, implying that bacteria sense mechanical stress and increase rotation in an attempt to escape from mucin gel. The last part of the thesis examines chemotaxis of H. pylori in presence of a linear pH gradient in broth using single-channel microfluidics. The chemotaxis of H. pylori was most prominent at pH3-4.5; below pH3 the bacteria is immotile and above pH4.5 the bacteria resume random swimming directions. Individual, directed trajectories suggest that bacteria slow down upon reaching pH5. Lastly, a microfluidic assay was developed to explore the viscous fingering of gastric acid in mucin. With proper injection pressure, single acid channels can be created in mucin solutions. Microrheology analysis reveals that mucin gels and swells in the vicinity of the acid channel, which squeezes the acid in forming a thin finger pattern.

abstract: Mechanical properties, in particular elasticity, of cancer cells and their microenvironment are important in governing cancer cell fate, for example function, mobility, adhesion, and invasion. Among all tools to measure the mechanical properties, the precision and ease of atomic force microscopy (AFM) to directly apply force—in the range of Pico to micronewtons—onto samples—with length scales from nanometers to tens of micrometers—has made it a powerful tool to investigate the mechanics of materials. AFM is widely used to measure deformability and stiffness of soft biological samples. Principally, these samples are indented by the AFM probe and the forces and indentation depths are recorded. The generated force-indentation curves are fitted with an elastic contact model to quantify the elasticity (e.g. stiffness). AFM is a precise tool; however, the results are as accurate as the contact model used to analyze them. A new contact model was introduced to analyze force-indentation curves generated by spherical AFM probes for deep indentations. The experimental and finite element analysis results demonstrated that the new contact model provides more accurate mechanical properties throughout the indentation depth up to radius of the indenter, while the Hertz model underestimates the mechanical properties. In the classical contact models, it is assumed that the sample is vertically homogenous; however, many biological samples—for example cells—are heterogeneous. A novel two-layer model was utilized to probe Polydimethylsiloxane hydrogel (PDMS) layers on PDMS substrates with stiffness mismatch. In this experiment the stiffness of the substrate was deconvoluted from the AFM measurements to obtain the stiffness of the layer. AFM and confocal reflectance microscopy were utilized along with a novel 3D microengineered breast cancer tumor model to study the crosstalk between cancer tumor and the stromal cells (CAFs) and the ECM remodeling caused by their interplay. The results showed that as the cancer cells invade into the extracellular matrix (ECM), they release PDGF ligands which enable Cafes to remodel the ECM and this remodeling increased the invasion rate of the cancer cells. Next, the effect of the ECM remodeling on anti-cancer drug resistant was investigated within the 3D microengineered cancer model. It was demonstrated that the combinatory treatment by anti-cancer and-anti-fibrotic drugs enhance the efficiency of the cancer treatment. A novel DNA-based 3D hydrogel model with tunable stiffness was investigated by AFM. The results showed the hydrogel stiffness can be enhanced by adding DNA crosslinkers. In addition, the stiffness was reduced to the control sample level by introducing the displacement DNA. Biophysical quantifications along with the in vitro microengineered tumor models provide a unique frame work to study cancer in more detail. / Dissertation/Thesis / Doctoral Dissertation Physics 2019

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Cells sense their environment and process changes through intracellular signaling networks to coordinate behavioral changes, such as cell fate decisions. In bacterial systems, these changes often occur over time periods longer than a single cell cycle. While we are now able to experimentally track and monitor these behavioral changes over multiple generations, we have a limited conceptual understanding of how these decisions are mediated by signaling pathways. Here, I present two projects that build predictive frameworks for understanding signaling pathway dynamics over multiple generations informed by the signal network architectures. In the first section, I use computational simulations to understand how signaling pathway architecture controls the duration over which related cells maintain similar concentrations of signaling pathway components following division from a common mother cell. I find that signal amplification is a requirement for similarity between related cells. In the second section, I take a joint theory-experiment approach to analyze the accumulation timescale of the signaling molecule cyclic di-GMP during biofilm initiation in the soil bacterium B. subtilis. Here I predict that the accumulation occurs over many generations, suggesting the possibility cyclic di-GMP is used as a cellular timer mechanism during biofilm initiation. These results both explain previous experimental findings as well as generate new predictions for how signaling pathways mediate single-cell behaviors in bacterial populations. Together, my work demonstrates the power of a joint theory-experiment approach to understand the long-term, dynamical behavior of intracellular signaling pathways by linking their architecture to their dynamical function.

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The focus of this research is multifunctional peptides, which interact with lipidbilayers as well as perform functions at the interface of the immune and nervous systems. Host survival is heavily dependent on molecules that adopt structures related to multiple functions. Studying the structure-function relationships of these peptides are helpful for discovering new ideas to treat diverse diseases, such as infectious diseases, neurological disorders, and inflammation. Two families of peptides are featured as archetypes: piscidin, a host defense peptide that was first isolated from the mast cells of hybrid striped bass and later reported to appear in neuroepithelial cells; orexin, which is able to activate G-protein coupled receptors that are involved in wakefulness and appetite, and participated in the host defense function. The first set of experiments focuses on Piscidin 1 (P1) and Piscidin 3 (3), which were optimized by evolution to display different but efficient potencies against pathogens despite being only 32% heterologous in sequence. The main mechanistic action of host defense peptides relies on the disruption of pathogenic membranes. Numerous mutants are designed to study not only the metal-carrying sequence of P1 and P3 but also the key structural differences between them. Half maximal effective concentration generated from dye leakage assays is used to quantify and compare the permeabilization capability of the peptides in relation to their membrane activity on pathogens. Membrane composition was chosen to reproduce bacteria dangerous to humans, including Escherichia coli and Vibrio species. P1 and P3 keep high activity against Vibrio species even they have the special ability to use polyunsaturated fatty acids to enhance virulence. Biological activity was characterized on these species by collaborations. The lipid oxidation assay was used to test our hypothesis that metallopeptide is able to generate reactive oxygen species and bring oxidative stress to the polyunsaturated fatty acids. On the structural side, circular dichroism was used to study the peptide secondary structure and establish the correlation between peptide α-helical content and membrane activity. Solid-state NMR is applied to obtain the high-resolution structures at the atomic level. In another set of experiments, the focus is on orexin A and its receptor. The attempt to synthesize the disulfide bonded peptide while also labeling for NMR is successfully attempted by a collaborator, enabling structural studies by CD and NMR. The receptor is cloned into bacteria and the expression tested, leading to promising data and paving the way for further research. In summary, we have investigated structure-function relationships in antimicrobial and neuroimmune peptides. This helps us better understand the diverse modes of action by host defense molecules in the immune and nervous systems, and thus may provide new ideas for developing peptide-based drugs for clinical applications.