abstract: In sub-Saharan Africa, an invasive form of nontyphoidal Salmonella (iNTS) belonging to sequence type (ST)313 has emerged as a major public health concern causing widespread bacteremia and mortality in children with malaria and adults with HIV. Clinically, ST313 pathovars are characterized by the absence of gastroenteritis, which is commonly found in “classical” nontyphoidal Salmonella (NTS), along with multidrug resistance, pseudogene formation, and chromosome degradation. There is an urgent need to understand the biological and physical factors that regulate the disease causing properties of ST313 strains. Previous studies from our lab using dynamic Rotating Wall Vessel (RWV) bioreactor technology and “classical” NTS strain χ3339 showed that physiological fluid shear regulates gene expression, stress responses and virulence in unexpected ways that are not observed using conventional shake and static flask conditions, and in a very different manner as compared to ST313 strain D23580. Leveraging from these findings, the current study was the first to report the effect of fluid shear on the pathogenesis-related stress responses of S. Typhimurium ST313 strain A130, which evolved earlier than D23580 within the ST313 clade. A130 displayed enhanced resistance to acid, oxidative and bile stresses when cultured in the high fluid shear (HFS) control condition relative to the low fluid shear (LFS) condition in stationary phase using Lennox Broth (LB) as the culture medium. The greatest magnitude of the survival benefit conferred by high fluid shear was observed in response to oxidative and acid stresses. No differences were observed for thermal and osmotic stresses. Based on previous findings from our laboratory, we also assessed how the addition of phosphate or magnesium ions to the culture medium altered the acid or oxidative stress responses of A130 grown in the RWV. Addition of either phosphate or magnesium to the culture medium abrogated the fluid shear-related differences observed for A130 in LB medium for the acid or oxidative stress responses, respectively. Collectively, these findings indicate that like other Salmonella strains assessed thus far by our team, A130 responds to differences in physiological fluid shear, and that ion concentrations can modulate those responses. / Dissertation/Thesis / Masters Thesis Microbiology 2017

Hypercholesterolemia is a significant risk factor in the development of cardiovascular disease and is associated with chronic leukocyte adhesion in the microvasculature. While the underlying mechanisms behind this have yet to be determined, it may be possible that hypercholesterolemia impairs the fluid shear stress (FSS) inactivation of neutrophils through the rigidifying effect of cholesterol on membrane fluidity. FSS restricts surface expression of CD18 integrins through cathepsin B (ctsB) proteolysis, which minimizes neutrophil adhesivity. If hypercholesterolemia blocks FSS mechanotransduction, then the inhibition of CD18 cleavage may link pathologic blood cholesterol elevations with dysregulated neutrophil adhesion. We hypothesized that elevated cholesterol contributes to dysregulated neutrophil adhesion by impairing ctsB FSS-induced CD18 cleavage through membrane fluidity changes. In the first part of this study, we demonstrated that FSS-induced CD18 cleavage is a robust response of neutrophils and involves selective cleavage of macrophage 1-antigen (Mac1) through ctsB proteolysis. The second part of this study confirmed that ctsB regulates neutrophil adhesion through its proteolytic actions on Mac1, an important integrin involved in adhesion and chemotaxis. Specifically, ctsB accelerated neutrophil motility through an effect on Mac1 integrins during pseudopod retraction. Furthermore, by using a flow-based assay to quantify the mechanoregulation of neutrophil adhesivity, we demonstrated that FSS-induced ctsB release promoted neutrophil detachment from platelet-coated substrates and unstimulated endothelium. For the third part of this study, we linked cholesterol-related membrane fluidity changes with the ability of FSS to restrict neutrophil adhesion through Mac1. We also determined that pathologic cholesterol elevations associated with hypercholesterolemia could block FSS-induced Mac1 cleavage and were linked to disrupted tissue blood flow. This was accomplished using low-density lipoprotein receptor deficient (LDLR-/-) mice fed a high-fat diet. Ultimately, the results provided in the present study confirmed that cholesterol-related changes in membrane fluidity blocked the ability of ctsB to regulate neutrophil adhesion through FSS-induced Mac1 cleavage. This implicates an impaired neutrophil FSS mechanotransduction response in the dysregulation of neutrophil adhesion associated with hypercholesterolemia. Since dysregulated adhesion may be one of the earliest upstream features of cardiovascular disease associated with hypercholesterolemia, the present study provides a foundation for identifying a new mechanobiological factor in the pathobiology of microcirculatory dysfunction.

Cancer cells traveling to distant tissues during metastasis must survive passing through the circulation. However, the influence of this fluid microenvironment on these cells is poorly understood. It was previously viewed that exposure to the hemodynamic shear forces within circulation was inhospitable to cancer cells, causing the cells to be destroyed. Recent evidence indicates that transformed cells are markedly more resistant to fluid shear stress when compared to non-transformed epithelial cells. Furthermore, these cells selectively adapt following exposure to fluid shear stresses and become more resistant to subsequent exposures to shear stress. The mechanisms behind this difference in phenotype and induced resistance are investigated. The elastic modulus, a measure of stiffness, may play a role in resistance and is shown to be altered upon exposure to fluid shear forces. Additionally, plasma membrane repair is a critical process in the resistance phenotype as cells sustain damage but are able to maintain viability. Cytoskeletal dynamics are also shown to play a role in resistance to fluid shear forces.

Ph.D. / Biomedical Engineering / The cardiovascular disease atherosclerosis is directly linked to the functions of the endothelium, the monolayer of endothelial cells (ECs) that line the lumen of all blood vessels. EC functions are affected by fluid shear stress (FSS), the tangential force exerted by flowing blood. In vivo FSS is determined by vascular geometry with relatively straight vessels producing high, unidirectional FSS and vessel branch points and curvatures producing low, oscillatory FSS. While these distinct FSS conditions differentially regulate EC functions, they also dramatically affect EC shape and cytoskeletal structure. High and unidirectional FSS induces EC elongation and cytoskeletal alignment, while concurrently promoting EC functions that are atheroprotective. In contrast, low and oscillatory FSS induces cobblestone-shaped ECs with randomly oriented cytoskeletal features, while simultaneously promoting EC functions that create an athero-prone vascular environment. Whether these distinct EC shapes and cytoskeletal structures influence EC functions, independent of FSS, is largely unknown. The overall hypothesis of this study is that cell shape and cytoskeletal structure regulate EC functions through mechanisms that are independent of FSS. Due to advances in surface engineering in the field of micropatterning, EC shape can be controlled independent of external forces by creating spatially localized surface cues. In this research, lanes of protein were micropatterned on glass surfaces to induce EC elongated shape in the absence of FSS. In Aim 1, micropattern-elongated EC (MPEC) shape and cytoskeletal structure were fully characterized and determined to be comparable to FSS-elongated ECs. Thus, inducing EC elongation on micropatterned lanes provides a platform for studying the functional consequences of EC shape, independent of FSS. Using this model, the following important markers of EC functions related to atherosclerosis were evaluated to determine the influence of EC shape and cytoskeletal alignment: extracellular matrix deposition (Aim 2), inflammatory function(Aim 3), and thrombotic potential (Aim 4). The results indicate that EC-elongated shape and cytoskeletal alignment participate in promoting selected EC functions that are protective against atherosclerosis, independent of FSS. Since EC shape is governed by the cytoskeleton, this data suggests that the cytoskeleton plays an active role in the regulation of EC functions that promote cardiovascular health.

Cardiovascular disease (CVD) is the leading cause of death internationally. Efforts to decrease CVD death has been explored through stem cell technology, specifically organoid formation. Current cardiac organoid models lack the vascular networks for nutrient supply and maturation. In this study, pillar perfusion technology is used to fabricate cardiac organoids and induce vascularization via dynamic culturing and the addition of vascular promoting growth factors (GFs). In addition to this study, a millifluidic chip is engineered for shear stress application via flow simulations and experimental flow analysis. We successfully optimized the millifluidic chip to achieve fluid shear stress of 20mPa and validated through particle tracking velocimetry using 0.1um diameter beads under flow. The results of cardiac organoids displayed contraction and growth of endothelial cells (ECs) under dynamic flow with GFs. In addition, smooth muscle cells (SMCs) displayed growth via GFs in both dynamic and static culturing. Although vascular networks were not present in all conditions of this experiment, this thesis can serve a basis for searching other methods of inducing vascularization.

Im 1997 wurden der Orphan GPR30 aus HUVECs kloniert, die FSS ausgesetzt waren. In dieser Studie konnte gezeigt werden dass die Expression von GPR30 durch die FSS-Behandlung im Vergleich zu unbehandelten HUVEC-Zellen deutlich induziert wurde. Daraufhin wurde in einer Studie von Isensse et al. die zelluläre und gewebsspezifische Expression von GPR30 in GPR30-LacZ Reportergen-Mäusen untersucht. Es konnte eine Expression von GPR30 vorwiegend in den Endothelzellen der kleinen Arterien verschiedenster Gewebetypen nachgewiesen werden. GPR30 war postuliert dass E2 direkt binden kann und dadurch rasche nicht-genomische Signale vermittelt. Im Gegensatz dazu haben verschiedene andere Veröffentlichungen gezeigt dass E2 nicht spezifisch an GPR30 bindet. Trotz der Kontroverse ob es sich bei GPR30 um einen Östrogenrezeptor oder nicht ist bislang nichts über seine Interaktion zu anderen Proteinen und deren Wechselwirkung bekannt. Deswegen war ein Ziel dieser Arbeit, Interaktionspartner von menschlichen GPR30 zu identifizieren und folglich ein humanes vaskuläres in vitro Modell zu etabliren, um die potentiellen Interaktionen von GPR30 sowie die downstream-Effekte der Wechselwirkung zwischen GPR30 und den neuen Interaktionspartner des vaskulären Modells auf Transkriptebene zu evaluieren. Ein Screening einer humanen kardiovaskulären cDNA-Bibliothek mit Hilfe des Y2H-Systems führte zur Identifizierung mehrerer Interaktionspartner für GPR30 darunter PATJ und FUNDC2. Durch anschließende CoIP konnte die Interaktion von GPR30 mit PATJ validiert werden. Des Weiteren konnte in dieser Arbeit die Wirkung von FSS auf die Expression von GPR30 in HUVECs bestätigt und ebenfalls in weiteren anderen Endothelzellen gezeigt werden. Abschließend wurde die Rolle von GPR30 und PATJ bei der Reaktion auf FSS auf transkriptioneller Ebene in HMEC-1-Zellen genomweit untersucht. Interessanterweise war eine Gruppe von Genen aufgrund von FSS in Zellen die GPR30 überexprimierten dereguliert als alleine durch FSS. / In 1997, the orphan G protein-coupled receptor 30, GPR30, was cloned using HUVECs exposed to FSS. It was shown that the level of GPR30 expression was up-regulated in response to FSS. Subsequently, in a study performed in the laboratory where the work for this thesis was carried out, the cellular and tissue distribution of GPR30 were investigated in GPR30-LacZ reporter mice and the expression was found predominantly in the endothelial cells of small arteries in several tissue types. GPR30, was also claimed to bind 17-β-estradiol (E2) directly and to mediate rapid non-genomic signalling. In contrast, various reports have indicated that E2 fails to bind GPR30 in a specific manner. Despite the controversy on whether GPR30 is an estrogen receptor or not, nothing is known at present about its relation and interaction with other proteins. Therefore, the aim of the work described in this thesis was to identify human GPR30 protein interaction partners and to establish a human vascular in vitro model in order to evaluate the potential role of GPR30 and the downstream effects of the interaction between GPR30 and new interaction partners in a vascular model at transcript level. The screening of a human heart cDNA library using the yeast two-hybrid assay led to the identification of several interaction partners for GPR30, among them PATJ and FUNDC2. These interactions were verified by CoIP experiments and the interaction of GPR30 with PATJ could be confirmed. The effect of FSS on the expression of GPR30 was confirmed in HUVECs and was detected in other endothelial cell types. In HUAECs, HAoECs and HMEC-1 cells GPR30 was also found up-regulated upon FSS, suggesting that GPR30 may indeed play a key role in vascular physiology. Finally, the role of GPR30 and PATJ in the FSS response was investigated at the genome-wide transcript level in HMEC-1 cells. Interestingly, a different panel of genes was deregulated owing to FSS in cells over-expressing GPR30 compared to FSS alone.

Osteocytes make up the largest cell population in bone and are believed to be the main mechanosensory bone cells. During mechanical disuse and overuse, osteocyte viability is compromised and is found to be co-localized with increased osteoclastic bone resorption. Osteoclasts are recruited to remodel sites of apoptosis or bone microdamage; however, it is unclear whether the apoptotic or neighbouring healthy osteocytes are responsible for targeted bone remodeling. I hypothesized that apoptotic osteocytes are: (a) directly responsible for initiating bone remodeling by recruiting osteoclast precursors and directing osteoclast differentiation, and (b) indirectly responsible by signaling to nearby healthy osteocytes that, in turn, regulate osteoclastogenesis. In this in vitro study, apoptotic osteocytes were found to increase osteoclast precursor migration and osteoclast formation. Inhibition of the osteoclastogenic protein, receptor activator of nuclear factor kappa B ligand (RANKL), in conditioned medium abolished the osteoclastogenic effect of apoptotic osteocytes. Healthy osteocytes surrounded by apoptotic regions were modeled by applying apoptotic osteocyte conditioned medium to healthy osteocytes. These cells also promoted osteoclastogenesis, and had increased expression of macrophage colony stimulating factor (M-CSF) and vascular endothelial growth factor (VEGF). Inhibition of these factors abrogated the pro-osteoclastic effect of healthy osteocytes conditioned by apoptotic osteocytes. These findings support the hypothesis that apoptotic osteocytes directly and indirectly, by signaling to nearby healthy osteocytes, initiate osteoclastogenesis. One limitation of our and other conventional in vitro models is the lack of real-time cell communication and physiologically-relevant mechanical environment. Using a microfluidics approach, a miniature fluid shear delivery system was created for in vitro osteocyte cultures. The purpose of this microsystem was to increase control of the cell microenvironment for subsequent integration into scalable screening platforms or co-culture systems for studying osteocyte mechanobiology under physiological loading conditions. Fluid shear stress was periodically applied without external pumping using a deflecting elastomer membrane, where up to 2 Pa of oscillating shear stress was possible by manipulating membrane dimensions. Osteocyte culture, viability and calcium response were demonstrated in the microdevice. Further studies should attempt to characterize calcium signaling in osteocytes which, using a conventional macro-scale system, was found to dependent on cell-cell communication.

Osteocytes make up the largest cell population in bone and are believed to be the main mechanosensory bone cells. During mechanical disuse and overuse, osteocyte viability is compromised and is found to be co-localized with increased osteoclastic bone resorption. Osteoclasts are recruited to remodel sites of apoptosis or bone microdamage; however, it is unclear whether the apoptotic or neighbouring healthy osteocytes are responsible for targeted bone remodeling. I hypothesized that apoptotic osteocytes are: (a) directly responsible for initiating bone remodeling by recruiting osteoclast precursors and directing osteoclast differentiation, and (b) indirectly responsible by signaling to nearby healthy osteocytes that, in turn, regulate osteoclastogenesis. In this in vitro study, apoptotic osteocytes were found to increase osteoclast precursor migration and osteoclast formation. Inhibition of the osteoclastogenic protein, receptor activator of nuclear factor kappa B ligand (RANKL), in conditioned medium abolished the osteoclastogenic effect of apoptotic osteocytes. Healthy osteocytes surrounded by apoptotic regions were modeled by applying apoptotic osteocyte conditioned medium to healthy osteocytes. These cells also promoted osteoclastogenesis, and had increased expression of macrophage colony stimulating factor (M-CSF) and vascular endothelial growth factor (VEGF). Inhibition of these factors abrogated the pro-osteoclastic effect of healthy osteocytes conditioned by apoptotic osteocytes. These findings support the hypothesis that apoptotic osteocytes directly and indirectly, by signaling to nearby healthy osteocytes, initiate osteoclastogenesis. One limitation of our and other conventional in vitro models is the lack of real-time cell communication and physiologically-relevant mechanical environment. Using a microfluidics approach, a miniature fluid shear delivery system was created for in vitro osteocyte cultures. The purpose of this microsystem was to increase control of the cell microenvironment for subsequent integration into scalable screening platforms or co-culture systems for studying osteocyte mechanobiology under physiological loading conditions. Fluid shear stress was periodically applied without external pumping using a deflecting elastomer membrane, where up to 2 Pa of oscillating shear stress was possible by manipulating membrane dimensions. Osteocyte culture, viability and calcium response were demonstrated in the microdevice. Further studies should attempt to characterize calcium signaling in osteocytes which, using a conventional macro-scale system, was found to dependent on cell-cell communication.

Vascularization of tissue-engineered substitutes is imperative for successful implantation into sites of injury. Strategies to promote vascularization within tissue-engineered constructs have focused on incorporating endothelial or endothelial progenitor cells within the construct. However, since endothelial and endothelial progenitor cells are adult cell types and limited in number, acquiring quantities needed for regenerative medicine applications is not feasible. Pluriopotent stem cells have been explored as a cell source for tissue-engineered substitutes because of their inherent ability to differentiate into all somatic cell types, including endothelial cells (ECs). Current EC differentiation strategies require laborious and extensive culture periods, utilize large quantities of expensive growth factors and extracellular matrix, and generally yield heterogenous populations for which only a small percentage of the differentiated cells are ECs. In order to recapitulate in vivo embryonic stem cell (ESC) differentiation, 3D stem cell aggregates or embryoid bodies (EBs) have been employed in vitro. In the developing embryo, fluid shear stress, VEGF, and oxygen are instructive cues for endothelial differentiation and vasculogenesis. Thus, the objective of this work was to study the effects of fluid shear stress pre-conditioning of ESCs on EB endothelial differentiation and vasculogensis. The overall hypothesis is that exposing ESCs to fluid shear stress prior to EB differentiation will promote EB endothelial differentiation and vasculogenesis. Pre-conditioning ESCs with fluid shear stress modulated EB differentiation as well as endothelial cell-like cellular organization and EB morphogenesis. To further promote endothelial differentiation, ESCs pre-conditioned with shear were treated with VEGF. Exposing EBs formed from ESCs pre-conditioned with shear to low oxygen resulted in increased production of VEGF and formation of endothelial networks. The results of this work demonstrate the role that physical forces play in modulating stem cell fate and morphogenesis.

This thesis research investigates the adhesion mechanisms of protein molecules to surfaces of biomaterials. New understanding in such adhesion mechanisms will lead to materials design and surface engineering in order to extend the lifespan of implants. The present research evaluates and analyzes the adhesive strength of proteins on pure High Density Polyethylene (HDPE), Single Wall Carbon Nanotube (SWCNT) enhanced HDPE composites, Ti-C:H coating and Ti6Al4V alloys (grade 2). The adhesive strength was studied through fluid shear stress and the interactions between the fluid and material surfaces. The adhesive strength of protein molecules was measured through the critical shear strength that resulted through the fluid shear stress. The effects of surface and material properties, such as roughness, topography, contact angle, surface conductivity, and concentration of carbon nanotubes on adhesion were analyzed. Research results showed that the surface roughness dominated the adhesion. Protein was sensitive to micro-scale surface roughness and especially favored the nano-porous surface feature. Results indicated that the unpurified SWCNTs influenced crystallization of HDPE and resulted in a nano-porous structure, which enhanced the adhesion of the protein onto a surface. Titanium hydrocarbon coating on silicon substrate also had a porous topography which enhanced its adhesion with protein, making it superior to Ti6Al4V.