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To date, protein adsorption is an unavoidable response to implanted biomaterials. When proteins interact with materials, adverse biological events such as thrombus formation and inflammation can occur and challenge device efficacy. Protein adsorption is influenced by various material and surface properties which can be modified in efforts to alter the protein-material interactions and the subsequent cellular response. There is a need for simple modifications of commonly used biomaterials and the effect of these modifications on (1) material properties (2) proteins and (3) cells is important to study. In this work, the effect of modifying polydimethylsiloxane (PDMS) and its interactions with fetuin-A are studied for potential immunomodulatory properties. PDMS modifications are achieved by altering the ratio of PDMS formulations to simply and effectively control elastic modulus, and by coating PDMS with polydopamine (PDA), a molecule commonly used as a bioglue. Surface characterization confirmed that altering the PDMS formulation changed the elastic modulus without affecting surface wetting properties. Minor changes in surface roughness via atomic force microscopy and surface chemistry via x-ray photoelectron spectroscopy were detected on some samples, and the deposition of PDA was confirmed. Protein adsorption studies provided quantitative and qualitative data on fetuin-A interactions. It was determined that fetuin-A adsorption was influenced by the PDMS formulations, and that the preferential adsorption changed when adsorbed from a competitive environment. Following modification of samples with adsorbed fetuin-A, the inflammatory effects of fetuin-A were investigated by measuring the concentration of pro- and anti-inflammatory cytokines in response to modified and unmodified samples. Data suggest that elastic modulus influences cytokine secretion at certain timepoints, a result of varied protein adsorption amounts and orientations in response to material stiffness. The addition of a PDA layer demonstrated the potentially cytokine mitigating effect of PDA cell interactions and protein immobilization when compared to unmodified PDMS samples. / Thesis / Master of Applied Science (MASc)

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Many of the materials used today for cardiovascular implants exhibit good bulk mechanical properties but fail to provide desirable surface properties for reducing thrombogenicity and promoting tissue integration. In fact, biological responses at the blood-material interface, including non-specific protein adsorption, coagulation, and platelet adhesion and activation significantly limit the use of currently available materials in many blood contacting applications. As our understanding of the biological responses to foreign materials has grown, so too has the potential for creating 'bioactive' materials capable of inducing and directing beneficial cellular processes. One promising technique for circumventing undesirable blood-biomaterial interactions involves seeding vascular endothelial cells (ECs) onto synthetic vascular grafts as a means of exploiting the physiological anticoagulant characteristics of the endothelium. Methods for improving cell retention on these constructs include immobilization of cell recognition motifs on the biomaterial surface in order to improve interactions between cells and the synthetic substrate. However, there remains the need to better understand the interactions between surface bound ligands and cells, and the role of linker molecule chemistry on ligand bioactivity and cellular response. In the current work, a novel method was optimized for modifying poly (dimethylsiloxane) (PDMS) with cell adhesion peptides tethered via a heterobifunctional allyl-, NSC-terminated polyethylene oxide (PEO) linker molecule. These novel surfaces combine the protein repellant property of PEO with the cell binding property of cell adhesion peptides. It was found that surfaces modified in this manner reduced protein adsorption to PDMS while increasing cell adhesion. Therefore the use of a generic PEO linker molecule was shown to be a very promising method of reducing non-specific protein interactions while maintaining ligand bioactivity. Silicone surfaces were also modified with diaminobutane (DAB) dendrimers in an attempt to increase the surface capacity for attachment of biomolecules and to compare the effect of surface peptide density with ligand mobility. Grafting cell adhesion peptides via surface bound dendrimers was found to increase the surface peptide density when compared to peptides grafted via the PEO spacer alone. However, cell adhesion was not significantly improved on the dendrimer-peptide modified surfaces compared to PDMS controls. This observation provides evidence that the properties of the linker molecule used for attachment of cell adhesion peptides to a biomaterial surface may be a critical factor in determining peptide bioactivity. In this case the peptides bound to the surface via the highly mobile linear PEO linker showed increased cell adhesion when compared to peptides linked via the rigid, highly branched dendrimer. It is therefore hypothesized that ligand mobility on a biomaterial surface may significantly influence ligand-cell receptor interactions to an even greater extent than surface peptide density. / Thesis / Master of Applied Science (MASc)

We present a theoretical framework based on an extension of Dynamical Density Functional Theory (DDFT) to describe the structure and dynamics of cells in living tissues and tumours. DDFT is a microscopic statistical mechanical theory for the time evolution of the density distribution of interacting many-particle systems. The theory accounts for cell pair-interactions, different cell types, phenotypes and cell birth and death processes (including cell division), in order to provide a biophysically consistent description of processes bridging across the scales, including the description of the tissue structure down to the level of the individual cells. Analysis of the model is presented for a single species and a two-species cases, the latter describing competition between a cancerous and healthy cells. In suitable parameter regimes, model results are consistent with biological observations. Of particular note, divergent tumour growth behaviour, mirroring metastatic and benign growth characteristics, are shown to be dependent on the cell pair-interaction parameters.

Blood vessels are critical for the delivery of oxygen and nutrients to all cells in the body. To properly function, blood vessels and their primary components must develop and mature into a healthy network, capable of dynamic alterations to meet new needs of the body. The early genetic and molecular programs that "push" the vasculature to develop are the same programs that reactivate when there are normal changes to the body such as injury, muscle growth or decline, or aging; and when pathologies arise like cancer, stroke, and diabetes. Therefore, it is crucial to understand how the vasculature develops into a healthy system by studying all components as they mature. Endothelial cells that comprise the vessels themselves are joined by specialized partner cells called pericytes that help guide and mature vessel growth. Pericytes lie elongated along endothelial cells and have multiple points of contact with the endothelium. In this position, pericytes assist in cell-cell communication and even blood flow regulation in the microvasculature. To study the relationship between endothelial cells and pericytes during development, we observed vascular morphology in three and four dimensions, as well as the genetic and molecular mechanisms underlying how these cells are recruited and interact in several experimental models. Thus, to thoroughly analyze the morphology of these vessels, we developed a rigorous methodology using a MATLAB program to determine the colocalization and coverage of pericytes associated with vessels in large image sets. After developing analytical methods to investigate all the components of the blood vessel wall, we expanded our investigation of how pericytes and other aspects of microvasculature develop in animal models, specifically a more commonly used murine model for vascular development and for treatment of human diseases. Our findings of vascular development in mice suggest that there are important differences in how human and mouse brain blood vessels form. Therefore, studies using mice must be carefully designed to account for these discrepancies. Additionally, research into why human and mouse neurovascular development and maturation are different can aid in the development of improved experimental models to better treat human pathologies. / Doctor of Philosophy / Blood vessels have the crucial job of delivering oxygen and nutrients to all the cells in the body. To perform this duty, blood vessels- and the components that make them- must develop and mature into a healthy network, capable of altering itself to meet new needs of the body. The early programs that “push” the vessel system to develop are the same programs that reactivate when there are normal changes to the body such as injury, muscle growth or decline, or aging; and when abnormal diseases arise like cancer, stroke, and diabetes. Therefore, it is critical to understand how blood vessels develop into healthy systems by studying all of their components as they mature. Endothelial cells that comprise the vessels themselves are joined by specialized partner cells called pericytes that help guide and mature vessel growth. Pericytes lie elongated along endothelial cells and have multiple points of contact with the endothelium. In this position, pericytes assist in cell-cell communication and even blood flow regulation in smaller vessels called capillaries. To study the relationship between endothelial cells and pericytes during development, we observed vascular anatomy in three and four dimensions, as well as mechanisms underlying how these cells come together and interact in several experimental models. Thus, to thoroughly analyze the morphology of these vessels, we developed a rigorous methodology using a MATLAB program to determine the colocalization and coverage of pericytes associated with vessels in large image sets. After developing analytical method to investigate all the components of the blood vessel wall, we expanded our investigation of how pericytes and other aspects of blood vessels develop in animal models, specifically a more commonly used animal model for vascular development and for treatment of human diseases. Our findings of vascular development in mice suggest that there are important differences in how human and mouse brain blood vessels form. Therefore, studies using mice must be carefully designed to account for these discrepancies. Additionally, research into why human and mouse neurovascular development and maturation are different can aid in the development of improved experimental models to better treat human illness and injury.

Alors que le rôle des cellules du système immunitaires innées sur la progression tumorale est l'objet d'une investigation croissante, le rôle des neutrophiles sur la sensibilité à la thérapie n'a pas été précédemment décrit. Jusqu’au présent, nous avons effectué des cocultures de neutrophiles et des différentes lignées cellulaires de lymphome non hodgkinien (LNH) en présence de divers agents cytotoxiques ou des thérapies ciblées. Afin d’évaluer l'effet du traitement sur la prolifération cellulaire et la mort des cellules, des marquages CFSE et DAPI ont été effectués respectivement, en utilisant la cytométrie en flux. Les neutrophiles ainsi que les cellules HL60 différenciées avec des propriétés de neutrophiles, ont atténué la sensibilité de cellules de lymphome à des agents anticancéreux in vitro, à la fois dans les modèles 2D et 3D. L'effet protecteur des neutrophiles a été testée in vivo en injectant des cellules de LNH et des neutrophiles chez des souris SCID/CB17 traités avec vincristine. La coinjection de neutrophiles réduit la sensibilité des cellules LNH à la chimiothérapie. Cet effet protecteur a été validé en utilisant des cellules primaires, purifiée à partir de patients atteints de leucémie lymphoïde chronique, exposés à des agents cytotoxiques ou des agents ciblés en présence de neutrophiles autologues. La protection par les neutrophiles est contact dépendante. Elle est médiée par l'interaction de CD11b et ICAM1, exprimé par les neutrophiles et les lymphocytes B, respectivement, et par la molécule d'adhésion CD44. Elle est également dépendante de Mcl1 et est partiellement abrogée par un composé anti-Mcl1 / While the role of innate immune cells on tumor progression is the object of increasing scrutiny, the role of neutrophils on sensitivity to therapy has not been previously described. To this end, we performed cocultures of freshly purified human neutrophils and different non- Hodgkin lymphoma (NHL) cell lines in the presence of various cytotoxic and targeted agents. CFSE and DAPI assays were performed to assess the therapeutic effect on cell proliferation and cell death, respectively, using flow cytometry. Neutrophils and differentiated HL60 cells with neutrophil-like properties attenuated the sensitivity of lymphoma cells to anti-cancer agents both in 2D and 3D models in vitro. The protective effect of neutrophils was tested in vivo using SCID/CB17 mice inoculated with NHL cells together with neutrophils, and treated with vincristine. The co-inoculation of neutrophils reduced the sensitivity of NHL cells to chemotherapy. Similar findings were made on primary cells, purified from patients diagnosed with chronic lymphocytic leukemia, exposed to cytotoxic agents or recently approved targeted agents (ibrutinib and idelalisib) in the presence of autologous neutrophils. Neutrophil-induced protection was dependent on cell-cell contact mediated by the interaction of CD11b and ICAM-1, expressed by neutrophils and B cells respectively, and by the adhesion molecule CD44. This protective effect was Mcl-1-dependent and was partially abrogated by an anti- Mcl-1 compound

Inflammation is a driving force behind various lethal pathological conditions, including atherosclerosis and cancer metastasis. Inflammatory mediators are keys that unlock the immune response via triggering interactions of leukocytes and vascular endothelial cells. Oxidized low density lipoprotein (OxLDL), lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-α), and histamine are all strong activators of the inflammatory response, and they can be simultaneously produced during various inflammatory disorders. This study focuses on investigating the cooperative effect of TNF-α and histamine, as well as OxLDL and histamine on monocyte-endothelium interactions. Furthermore, the roles of LPS-activated monocytes and histamine in interactions of breast cancer cells with vascular endothelium are also explored. The results suggest that: 1) TNF-α and histamine have a synergistic effect on monocyte-endothelium interactions; 2) histamine cooperatively works with OxLDL to induce monocyte recruitment on endothelial cells; 3) OxLDL treated macrophages and mast cells respectively release TNF-α and histamine, which in turn synergistically increase the capture of monocytes by endothelial cells; 4) LPS activated monocytes secret TNF-α to activate endothelial cells and assist in adhesion of carcinoma cells to activated vascular endothelium; 5) exposure of activated endothelial cells to histamine exaggerates the carcinoma cell arrest, especially in the presence of LPS activated monocytes. In summary, this study implies that an increased risk of atherogenesis may be among the population affected by allergy or asthma and having lipid rich diet that leads to the production of TNF-α and OxLDL in body. It also highlights that the mechanism by which Gram-negative bacterial infection increases a risk of breast cancer metastasis through the cardiovascular system, and shows the necessity to develop better approaches for preventing this infection after surgical resection or before histamine combined IL-2 immune therapy in order to reduce a risk of cancer metastasis. / acase@tulane.edu

The typical response of cancer patients to treatment is only temporary, and is often followed by relapse. The failure of various therapeutic strategies is commonly attributed to the emergence of drug resistance. The response patterns for patients under such treatments indicate that complex dynamics regulate the response of the tumour to the therapy. The environment in which the tumour lives (the stroma) is known to be a modulator of multiple mechanisms that lead to drug resistance and seems to be a likely candidate for explaining some of this complexity. Understanding the role of stromal cells in the promotion of drug resistance is critical for the design of optimal treatment strategies, and for the development of novel therapies that selectively target both the tumour and the stroma. In this thesis we design two novel mathematical models that describe cancer growth within its environment and the evolution of drug resistance within spatially complex and temporally dynamic tumours. A compartment model captures clinically observed dynamics and allows direct comparison with experimental data, facilitating model parametrisation and the understanding of inter-tumour heterogeneity. An individual cell-based model highlights the key role of local interactions, determining heterogeneity at the tissue scale, that will eventually determine treatment outcome. A non-spatial approximation of this second model allows us to find analytic guidelines for the design of effective therapy. These tools allow the simulation of a range of treatment strategies (including combination of different drugs and variation of schedule) as well as the investigation of therapy response based on patient- or organ-specic parameters. The work developed in this dissertation is based on the paradigmatic biology of melanoma and non-small cell lung cancer. Its results are therefore applicable to a variety of cancer treatments that target similar processes, and whose therapeutic failure can be attributed to environment-mediated drug resistance.