Insights from modeling metabolism and amoeboid cell motility in the immune system

This thesis focuses on two processes involved in fighting infections: metabolism and immune cell motility and navigation.Regarding metabolism, we present ZebraGEM 2.0, an improved whole-genome scale metabolic reconstruction for zebrafish, that we used to study zebrafish metabolism upon infection with Mycobacterium marinum integrating gene expression data from control and infected zebrafish larvae. The chapters focusing on cell motility in response to the environment, revolve around the question of how the environmental inputs of cell-matrix interactions, cell-sized obstacles and cell-signalling upon wounding shape and guide cell motility.Analysis and Stochastic

Tracking and analysis of movement at different scales: from endosomes to humans

Movement is apparent across all spatio-temporal scales in biology and can have a significant effect on the survival of the individual. For this reason, it has been the object of study in a wide range of research fields, i.e. in molecular biology, pharmaceutics, medical research but also in behavioural biology and ecology. The aim of the thesis was to provide methodologies and insight on the movement patterns seen at different spatio-temporal scales in biology; the intra-cellular, the cellular and the organism level. At the intra-cellular level, current thesis studied the compartmental inheritance in Human Osteosarcoma (U2-OS) cells. The inheritance pattern of the endosomal quantum dot fluorescence across two consecutive generations was for first time empirically revealed. In addition, a in silico model was developed to predict the inheritance across multiple generations. At the cellular level, a semi-automated routine was developed that can realize long-term nuclei tracking in U2-OS cell populations labeled with a cell cycle marker in their cytoplasm. A method to extract cell cycle information without the need to explicitly segment the cells was proposed. The movement behaviour of the cellular population and their possible inter-individual differences was also studied. Lastly, at the organism level, the focus of the thesis was to study the emergence of coordination in unfamiliar free-swimming stickleback fish shoals. It was demonstrated that there exist two different phases, the uncoordinated and the coordinated. In addition, the significance of uncoordinated phase to the establishment of the group’s social network was for first time evinced. The adaptation of the stickleback collectives was also studied over time, i.e. the effect of group’s repeated interactions on the emergence of coordination. Findings at the intra-cellular and cellular level can have significant implications on medical and pharmaceutical research. Findings at the organism level can also contribute to the understanding of how social interactions are formed and maintained in animal collectives

Computational methods for investigating cell motility with applications to neutrophil cell migration

Cell motility is closely linked to many important physiological and pathological events such as the immune response, wound healing, tissue differentiation, embryogenesis, in ammation, tumour invasion and metastasis. Understanding the ability of cells to alter their shape, deform and migrate is of vital importance in many biological studies. The rapid development in microscopy and imaging techniques has generated a huge amount of discrete data on migrating cells in vivo and in vitro. A key challenge is the use of discrete experimental observations to develop novel methods and algorithms that track cells and construct continuous trajectories of their motion as well as characterising key geometric quantities associated with cell migration. Therefore, in this work using robust numerical tools we focus on proposing and implementing mathematical methodologies for cell movement and apply them to model neutrophil cell migration. We derive and implement a computational framework that encompasses modelling of cell motility and cell tracking based on phase field and optimal control theory. The cell membrane is represented by an evolving curve and approximated by a diffuse interface; while the motion of the cell is driven by a force balance acting normal on the cell membrane. This approach allows us to characterise the locus of the centroid cell-surface position. In addition, we describe a surface partial differential equation framework that can be coupled with the phase-field framework, thereby offering a wholistic approach for modelling biochemical processes and biomechanics properties associated with cell migration

Biophysical Aspects of Leukocyte Transmigration through the Vascular Endothelium

Leukocyte transmigration through the vascular endothelium is a key step in the immune response, and also in progression of the cardiovascular disease atherosclerosis. Much work has previously focused on the biological aspects of leukocyte transmigration, such as cytokine exposure, junctional protein organization in the endothelium, and signaling pathways. However, in recent years, many studies have identified links between the mechanical properties of the cellular microenvironment and cell behavior. This is relevant to the cardiovascular system in two ways: (1) it is likely that the mechanical properties of vasculature depend on both vessel size (large vessels versus microvasculature) and tissue type (soft brain versus stiffer muscle or tumor), and (2) both large vessels and microvasculature stiffen in atherosclerosis. For the first time, this dissertation provides a quantitative evaluation of the biophysical effects of vasculature stiffening on endothelial cell (EC) biomechanical properties, as well as leukocyte migration and transmigration. A novel in vitro model of the vascular endothelium was created. This model mimics physiological conditions more closely than previous models, by taking into account the flexibility of the subendothelial matrix; previous models have mostly utilized glass or plastic substrates that are much stiffer than physiological. EC monolayers were formed on extracellular matrix (ECM) protein-coated hydrogels and activated with tumor necrosis factor-α or oxidized low density lipoprotein to induce an inflammatory response. We determined that three important components of the in vitro model (cell-cell adhesion, cytokine exposure, and subendothelial matrix stiffness) have significant effects on EC biomechanical properties. Next, we showed that neutrophils are mechanosensitive, as their migration is biphasic with substrate stiffness and depends on an interplay between substrate stiffness and ECM protein amount; these results suggest that any biomechanical changes which occur in vasculature may also affect the immune response. Finally, we discovered that neutrophil transmigration increases with subendothelial matrix stiffness, and we demonstrated that this effect is due to substrate stiffness-dependent EC contractile forces. These results indicate, for the first time, that the biophysical states of the endothelium and subendothelial matrix, which likely vary depending on size, location, and health of vasculature, are important regulators of the immune response

The Role of Red Blood Cell Membrane Rigidity on Cellular and Drug Particle Carrier Dynamics in Blood Flow

Blood and heart-related diseases remain a significant challenge for modern-day medicine. Blood cell-related diseases have also proven to be challenging to understand and treat, specifically diseases involving the loss of deformability (rigid) in red blood cells (RBCs). Disease involving rigid RBCs are typically of genetic origin and thus limit treatment options and treatment efficacy. Rigid RBC disorders give rise to many medical complications, including vaso-occlusion, pulmonary hypertension, and cardiac dysfunction. Patients inflicted with Sickle Cell Disease (SCD), hereditary spherocytosis, iron-deficient anemia, pyruvate kinase deficiency, human immunodeficiency virus (HIV), malaria, sepsis, and even natural aging all have less deformable (rigid) RBCs than healthy patients. Rigid RBCs cause major physical damage when traveling through the body by occluding microvasculature, depriving tissues of nutrients, and damaging walls of the spleen, liver, and lungs. The core work presented in this dissertation aims to probe how decreases in RBC deformability affect hemodynamics and impact functionality of other blood cells, clarifying the pathology of RBC-related diseases. We initially present a model of artificially rigidified human RBCs which offers an experimental control over extent of membrane stiffness as well as the fractional composition of rigid RBCs in whole blood. Here, we find that the presence of rigid RBCs in blood flow significantly alters the ability of immune cells to adhere to inflammation on the vascular wall of a microfluidic model. In some cases, the presence of highly rigid RBCs reduces leukocyte adhesion to the vascular wall by up to ~80%. Following this initial investigation, we take a pivotal focus on SCD and further quantifying the whole blood characteristics of SCD pediatric patient blood and its behavior in flow. This thesis presents multiple investigations highlighting the outcome of RBC rigidity in SCD. An interesting clinical case study is highlighted in this work as well as additional in vitro work showing how the presence of RBC rigidity alters immune cell adhesion functionality. An artificial model of blood infusion therapy is also developed to test how leukocyte adhesion to inflammation is impacted upon alteration of whole blood composition. This knowledge is essential in understanding why people with diseases related to RBC deformability are susceptible to infection and have irregular immune responses. In addition, we also investigate how rigid RBCs in blood flow alter the adhesion efficacy of vascular-targeted carriers (VTCs). The field of drug delivery has taken an interest in combating numerous blood and heart diseases such as atherosclerosis via the use of VTCs. Ideally, VTC technology increases drug delivery efficacy and simultaneously reduces cytotoxic effects, precisely localizing drugs only to the disease site through receptor-ligand interactions. Cellular interactions are not yet fully understood. The dynamics of disease-inflicted cells (rigid RBCs) are even less understood, thus compounding the problem of efficient VTC design under diseased blood conditions. We investigate various particle design parameters and assess their vascular wall adhesion performance in the presence of rigid RBCs. We find the vascular adhesion of stiff microparticles is reduced by up to ~50% in the presence of rigid RBCs. Interestingly, deformable hydrogel microparticles can experience an increase in vascular adhesion of up to ~ 80%. This work explores an opportunity to develop new therapeutics with high efficacy in diseased blood.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/169853/1/gutieman_1.pd

Manipulation of Cell and Particle Trajectory in Microfluidic Devices

Microfluidics, the manipulation of fluid samples on the order of nanoliters and picoliters, is rapidly emerging as an important field of research. The ability to miniaturize existing scientific and medical tools, while also enabling entirely new ones, positions microfluidic technology at the forefront of a revolution in chemical and biological analysis. There remain, however, many hurdles to overcome before mainstream adoption of these devices is realized. One area of intense study is the control of cell motion within microfluidic channels. To perform sorting, purification, and analysis of single cells or rare populations, precise and consistent ways of directing cells through the microfluidic maze must be perfected. The aims of this study focused on developing novel and improved methods of controlling the motion of cells within microfluidic devices, while simultaneously probing their physical and chemical properties. To this end we developed protein-patterned smart surfaces capable of inducing changes in cell motion through interaction with membrane-bound ligands. By linking chemical properties to physical behavior, protein expression could then be visually identified without the need for traditional fluorescent staining. Tracking and understanding motion on cytotactic surfaces guided our development of new software tools for analyzing this motion. To enhance these cell-surface interactions, we then explored methods to adjust and measure the proximity of cells to the channel walls using electrokinetic forces and 3D printed microstructures. Combining our work with patterned substrates and 3-dimensional microfabrication, we created micro-robots capable of rapid and precise movements via magnetic actuation. The micro-robots were shown to be effective tools for mixing laminar flows, capturing or transporting individual cells, and selectively isolating cells on the basis of size. In the course of development of these microfluidic tools we gained valuable new insights into the differences and limitations of planar vs. 3D lithography, especially for fabrication of magnetic micro-machines. This work as a whole enables new mechanisms of control within microfluidics, improving our ability to detect, sort, and analyze cells in both a high throughput and high resolution manner

Quantifying Adhesion and Morphological Dynamics of Human Hematopoietic Stem and Progenitor Cells on Novel In Vitro Models of Bone Marrow Niche

Since both healthy human hematopoietic stem and progenitor cells (HSPC) and leukemia initiating cells (LIC) are sustained in a dormant state in bone marrow niche, they are protected against cytotoxic effects of chemotherapy. Thus, quantitative identification of differential adhesion of HSPC vs. LIC to bone marrow niche would help for the development of an effective clinical therapy of leukemia. The main aim of the present thesis was the fabrication and application of self-assembled, planar phospholipid membranes on solid support as in vitro model of bone marrow niche. A special focus was put on the influences of relevant ligand-receptor pairs and acute myeloid leukemia (AML) on the adhesion and morphological dynamics of HSPC. As the model of bone marrow niche, supported lipid membranes functionalized with N-cadherin and SDF1alpha were utilized to study their relative significance. In Chapter 2, the deposition of supported membranes and their quantitative functionalization with N-cadherin and SDF1alpha were confirmed by high energy specular X-ray reflectivity (XRR) and quartz crystal microbalance with dissipation monitoring (QCM-D). The fine structures perpendicular to the membrane surface and the lateral density of membrane-anchored proteins were determined by XRR with sub-Ångström resolution. Real-time monitoring by QCM-D of membrane deposition and functionalization demonstrated the quantitative variability of the average intermolecular distance of proteins and elucidated their viscoelastic properties such as the shear elastic modulus and shear viscosity. In Chapter 3, the strength of HSPC adhesion was quantitatively evaluated by the determination of (a) the fraction of adherent cells, (b) the area of tight adhesion and (c) the critical force of cell detachment as a function of the average intermolecular distance of N-cadherin nd SDF1alpha. The results clearly demonstrated that the binding of HSPC to the in vitro niche model was a positively cooperative process, and the adhesion mediated by the SDF1alpha/CXCR4 axis was stronger compared to adhesion mediated by the homophilic N-cadherin axis. The statistical image analysis of stochastic morphological dynamics unraveled that HSPC on in vitro niche models displaying SDF1a dissipated energy by undergoing oscillatory deformation, whereas cell locomotion mediated by the homophilic binding of N-cadherin was hardly impaired with morphological deformations. In order to verify the clinical relevance, the adhesion of leukemic blasts (LB) from AML patients was investigated in a systematic manner. In comparison to HSPC, LB exhibited a significantly higher affinity to the in vitro niche model reflecting the partial ineffectiveness of chemotherapy and the difficulties of replacing them by allogenic transplanted HSPC. The obtained results demonstrated that the combination of precisely defined cell surface models, a novel non-invasive assay for evaluating the cell adhesion strength, and statistical analysis of live cell images in Fourier space is a powerful tool to quantitatively analyze different functions of ligand-receptor pairs in bone marrow niche, which cannot be assessed by phenomenological observation