Nuclear Envelope Composition Determines The Ability Of Neutrophil-Type Cells To Passage Through Micron-Scale Constrictions

Neutrophils are characterized by their distinct nuclear shape, which is thought to facilitate the transit of these cells through pore spaces less than one-fifth of their diameter. We used human promyelocytic leukemia (HL-60) cells as a model system to investigate the effect of nuclear shape in whole cell deformability. We probed neutrophil-differentiated HL-60 cells lacking expression of lamin B receptor, which fail to develop lobulated nuclei during granulopoiesis and present an in vitro model for Pelger-Huët anomaly; despite the circular morphology of their nuclei, the cells passed through micron-scale constrictions on similar timescales as scrambled controls. We then investigated the unique nuclear envelope composition of neutrophil-differentiated HL-60 cells, which may also impact their deformability; although lamin A is typically down-regulated during granulopoiesis, we genetically modified HL-60 cells to generate a subpopulation of cells with well defined levels of ectopic lamin A. The lamin A-overexpressing neutrophil-type cells showed similar functional characteristics as the mock controls, but they had an impaired ability to pass through micron-scale constrictions. Our results suggest that levels of lamin A have a marked effect on the ability of neutrophils to passage through micron-scale constrictions, whereas the unusual multilobed shape of the neutrophil nucleus is less essential

Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cellfree cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development

Digital Microfluidics for Multidimensional Biology

Digital microfluidics (DMF) has emerged in the past decade as a novel microfluidic paradigm. As a liquid handling technology, DMF facilitates the electrostatic manipulation of discrete nano- and micro- litre droplets across open electrode arrays providing the advantages of single sample addressability, automation, and parallelization. This thesis presents DMF advances toward improved functionality and compatibility for automated miniaturized cell culture in two and three dimensions. Through the development and integration of surface patterning techniques we demonstrate a virtual microwell method for high precision on-device reagent dispensing in one and two plate DMF geometries. These methods are shown to be compatible with two-dimensional culture of immortalized cell lines on ITO, primary cells on coated surfaces, and for co-culture assays. We further extrapolate this method for the formation of microgels on-demand where form micro scale hydrogel structures through passive dispensing in a wide array of geometries. With this system we interrogate three-dimensional cell culture models, specifically for the recapitulation of kidney epthelialization and the analysis of functional cardiac microgels.Ph

Inhibition of bacterial adhesion to biomaterials by cranberry derived proanthocyanidins

Nosocomial, or hospital acquired, infections, are ubiquitous within the modern clinical setting leading to over $5 billion annually of related healthcare costs in North America. All indwelling devices are highly susceptible to bacterial colonization where physico-chemical interactions between bacteria and biomaterial surfaces have been implicated as determinant factors in the fate of the initial adhesion processes. It has been proposed that by exploiting interference strategies within this critical step of infection the ability to create 'non-infective' biomaterials may be developed.This thesis demonstrates the effectivity of North American cranberry (Vaccinium macrocarpon) derived proanthocyanidins in preventing the adhesion of pathogenic bacteria to biomaterial surfaces. Specifically, using a model of catheter associated urinary tract infection, significant reductions in initial adhesion of uropathogenic Escherichia coli and Enterococcus faecalis to PVC and PTFE were observed. With the application of colloidal theory, a mechanism of steric interference was determined as responsible for these effects.The evidence presented implicates PAC as a molecule of interest for the development of novel biomaterials with increased resistance to bacteria colonization

Transport of selected bacterial pathogens in agricultural soil and quartz sand

The protection of groundwater supplies from microbial contamination necessitates a solid understanding of the key factors controlling the migration and retention of pathogenic organisms through the subsurface environment. The transport behavior of five waterborne pathogens was examined using laboratory-scale columns packed with clean quartz at two solution ionic strengths (10 mM and 30 mM). Escherichia coli O157:H7 and Yersinia enterocolitica were selected as representative Gram-negative pathogens, Enterococcus faecalis was selected as a representative Gram-positive organism, and two cyanobacteria (Microcystis aeruginosa and Anabaena flos-aquae) were also studied. The five organisms exhibit differing attachment efficiencies to the quartz sand. The surface (zeta) potential of the microorganisms was characterized over a broad range of pH values (2–8) at two ionic strengths (10 mM and 30 mM). These measurements are used to evaluate the observed attachment behavior within the context of the DLVO theory of colloidal stability. To better understand the possible link between bacterial transport in model quartz sand systems and natural soil matrices, additional experiments were conducted with two of the selected organisms using columns packed with loamy sand obtained from an agricultural field. This investigation highlights the need for further characterization of waterborne pathogen surface properties and transport behavior over a broader range of environmentally relevant conditions