Biochemical and mechanical properties of the extracellular matrix (ECM) are known to independently influence cell function. Given the complexity of cellular responses, I hypothesized that the integration of multiple matrix factors as opposed to their individual contribution is key to understanding and controlling cell function. The objective of this thesis was to systematically investigate matrix biochemical and mechanical regulation of multipotent stromal cell (MSC) osteogenesis. First, I demonstrated that substrate stiffness-dependent MSC spreading, proliferation and osteogenic response were differentially regulated by matrix protein type (collagen I vs. fibronectin) and concentration. Second, I developed and characterized a matrix microarray platform that enabled the efficient screening of multiple matrix-derived cues (substrate stiffness, ECM type and density). I implemented the matrix microarray platform together with parametric regression models to elucidate novel matrix interactions in directing mouse MSC osteogenic and adipogenic differentiation. Third, I extended the screening study to examine matrix-dependent human MSC osteogenesis. Non-parametric regression models were used to provide a nuanced description of the multi-factorial matrix regulation in MSC osteogenesis. The response surfaces revealed a biphasic relationship between osteogenesis and substrate stiffness, with the exact location and magnitude of the optimum contingent on matrix composition. Guided by the screening results and perturbation to key cytoskeletal regulators, I identified a novel pathway involving Cdc42 in matrix mechanical and biochemical regulation of MSC osteogenesis. Surprisingly, Cdc42 mediated stiffness-dependent MSC osteogenesis independent of ROCK, suggestive of a contractility-independent mechanism in matrix rigidity signal transduction. In summary, the integration of cell-based arrays and statistical modeling has enabled the systematic investigation of complex cell-matrix interactions. This generalizable approach is readily adaptable to other cellular contexts, complementing hypothesis-driven strategies to facilitate non-intuitive mechanistic discovery. Moreover, the improved understanding of matrix-dependent MSC function also has practical relevance to the development of biomaterials for tissue engineering applications.

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Made available in DSpace on 2014-06-11T19:32:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-07-30Bitstream added on 2014-06-13T19:43:54Z : No. of bitstreams: 1 macedo_mls_dr_botfm_prot.pdf: 3586847 bytes, checksum: 528671d32509cb0d8b738be7fcab29b3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A hipertensão gestacional está presente em cerca de 10% das gravidezes e ainda é a primeira causa de mortalidade materna no Brasil. O diabetes gestacional complica 7,6% das gestações no Brasil e está associado a esultados perinatais insatisfatórios. Estas complicações cursam com disfunção endotelial e alteração da elasticidade da parede -.vascular. A onometria de aplanação é um método não invasivo, portátil e de fácil aprendizagem que avalia a função endotelial através do estudo da rigidez arterial (perda da elasticidade arterial). Além de avaliar a função endotelial este método oferece estudo indireto de vários parâmentros cardiovasculares centrais. O grande número de informações que este método obtém de maneira não invasiva, faz deste, um instrumento valoroso em pesquisa. Apresenta grande potencial, especialmente, na compreensão dos mecanismos fisiopatológicos que cursam com comprometimento vascular na gravidez. / Gestational hypertension affects 10% of pregnancies and is still the first :ause of maternal mortality in Brazil. Gestational diabetes affects 7,6% of gnancies in Brazil and is associated with an unsatisfactory peri-natal come. These complications are associated to endothelial dysfunction and abnormal elasticity of the arterial wall. Applanation tonometry is a nonvasive, portable and easy learning method that evaluates endothelial nction by the study of arterial stiffness (Iost of arterial elasticity). Beyond e endothelial function evaluation, this method gives, indirectly, several central cardiovascular parameters. The great number of information btained non invasively by this method, makes of this, a valuable instrument in research. It has special potential to help in the comprehension of the mechanisms of those diseases that presents with vascular commitment in pregnancy.

Glomerular podocytes are epithelial cells that are attached to outer glomerular basement memberane (GBM) by foot processes, and blood filtration occurs through podocytes, GBM, and endothelial cells. Podocytes are under constant mechanical stress due to their location around outside of glomerular capillaries, which can be associated with glomerular hypertension. It is important for podocytes to maintain their mechanical integrity, since podocyte adhesion to GBM is crucial to prevent podocyte loss, detachment, and associated alteration in cell adhesive properties, and further progression of glomerular disease. In this study, we examined the role of stiffness in podocyte function with hypothesis that increasing substrate stiffness would promote development of cell structural features that are associated with stronger adhesion. In order to test this, polyacrylamide substrates with different stiffness ranged from 3750 Pa to 152600 Pa were generated and immortalized mouse podocytes were cultured on these substrates. Then we measured how substrate stiffness affects cell morphology and several structural proteins distribution. We found that the size and the number of attached cells increased with longer actin filaments as stiffness of substrate increased. Since proteinuria or glomerulosclerosis can be associated with podocyte actin cytoskeleton defect, we suggest podocytes in a "softer" environment are vulnerable to glomerular diseases, since stress fibers were shorter and less organized as substrates decreased stiffness. Our results relating to the presence and distribution of certain proteins in cells were somewhat inconclusive, since intensity of synaptopodin and vinculin did not correspond to the changes of stiffness, due to the possibility of other underway mechanisms that interfere with podocyte adhesion. There was no clear relationship between YAP and the changes of substrate stiffness, and one possible explanation could be due to the optical irregularities in the substrate. Overall, this study was able to show that increased substrate stiffness promoted cell structural feature development in podocytes. However, further studies are needed to better understand how changes in substrate mechanical properties can affect structural protein distribution in these cells.

The physical characteristics (i.e., nanostructure and stiffness) of the extracellular matrix where cells reside have been shown to profoundly affect numerous cellular events in vivo and also been employed to modulate cell behavior in vitro, yet how these physical cues regulate cell behavior is still elusive. Therefore, we engineered a variety of nanotopographies with different shapes and dimensions, and investigated how the nanotopographical cue, through focal adhesions-cytoskeleton-nucleus pathway, affected cell phenotype and function. We further designed and fabricated well-defined substrates which had either identical biochemical cue (adhesive ligand presentation) but different nanotopographical cues or identical nanotopography but different biochemical cues, and dissected the roles of these cues in cell modulation. In addition, we revealed that the human mesenchymal stem cells (hMSCs) could obtain nanotopographical memory from the past culture environment, and the nanotopographical memory influenced the future fate decision of the hMSCs. Moreover, we evaluated the effects of substrate nanotopographical and stiffness cues on the fibrogenesis of human lung fibroblasts in response to carbon nanotubes and highlighted the significance of these physical cues in the development of physiologically relevant in vitro models for nanotoxicological study. The mechanistic understanding of the physical regulation of cell behaviors will provide important insight into the advancement of cell culture technologies and the recreation of biomimetic in vitro tissue/organ models.

A parametric study was performed to determine the effect of flexible foundations on the response of timber shear walls. Timber shear walls, which typically consist of structural-use panels, such as plywood or oriented strand board (OSB), attached to a frame made from dimension lumber with dowel-type fasteners such as nails, provide resistance to lateral loading for many low-rise structures in North America. Research performed on shear walls has assumed that a wall is supported by a relatively stiff foundation, such as a concrete block wall, along the entire length of the wall. However, walls are sometimes supported by a relatively flexible foundation, such as a floor joist, which would alter the stiffness, and therefore the response of the wall. Research on flexible foundations is limited at best, and there is a string need to examine the behavior of shear walls on flexible foundations. The study consisted of creating a shear wall numerical model, varying the conditions at the foundation of the model, and analyzing the model when subjected to both monotonic and dynamic loading for each foundation. The system modeled corresponded to a 2.4 m (8 ft) high by 3.7 m (12 ft) long shear wall supported by and parallel to a 7.3 m (24 ft) long joist with hold-downs at each chord of the wall. The joist was supported at each end, with one chord of the wall at an end of the joist and the other chord located at the center of the joist. Eleven joist cross-sections, with sizes determined based on deflection criteria ranging from L/180 to L/720, and a rigid base were included in the study, along with three different hold-down bolt sizes, for a total of thirty-six different foundations. The wall model was analyzed using WALSEIZ1, which is a modified version of the finite element program WALSEIZ (White and Dolan, 1995). Maximum displacements, internal forces, and maximum load were recorded when the model was subjected to monotonic loading, while the maximum displacements and base shear were recorded when the model was subjected to dynamic loading. Results from the study were examined to determine if modifications to the current design practices should be considered. / Master of Science

For more than half a century cardiovascular disease has been the leading cause of death in the United States.  Aging, hypertension, and obesity are major risk factors for cardiovascular disease and clearly associated with arterial stiffness.  Arterial stiffness generates higher afterloads and diminishes coronary perfusion thereby causing ventricular hypertrophy and ischemia.  Importantly, arterial stiffness is an independent predictor of cardiovascular disease risk and all-cause mortality.   Current strategies such as inhibition of angiotensin II or angiotensin converting enzyme, reduction of smooth muscle tone, blood volume, or inflammatory mediators, and improving glucose homeostasis are effective destiffening options.  Nebivolol, a third generation beta-blocker, has unique vasodilatory characteristics and may be particularly efficacious as a destiffening agent.  Only a few studies have addressed this issue while relying on indirect, blood pressure-dependent stiffness indices precluding clear understanding of study outcomes.  There remains a need to determine the potential utility of nebivolol therapy as an arterial destiffening strategy.   Thus, we hypothesized that the combination of nebivolol and lifestyle modification would reduce central arterial stiffness in middle-aged and older hypertensive adults more than either intervention alone.  To test this hypothesis, we randomized 45 hypertensive adults to receive lifestyle modification, nebivolol, or combination for 12 weeks.  β-stiffness index, pulse wave analysis, and arterial compliance were measured at baseline and following the intervention.  No baseline differences in variables of  interest were observed between groups.  In contrast to our hypothesis, lifestyle modification, nebivolol, and combination groups had similar (P>0.05) reductions in beta-stiffness index (-1.87±0.83; -2.03±0.60; and -2.51±0.90 U), respectively, while carotid-femoral pulse wave velocity declined only in the nebivolol and combination groups.   Our findings suggest combination of nebivolol and lifestyle modification reduces arterial stiffness to a similar degree as either intervention alone in middle-aged and older hypertensive adults.  Further studies are needed to determine if the changes in arterial stiffness continue to occur or remain clinically significant over longer durations. / Ph. D.

Indiana University-Purdue University Indianapolis (IUPUI) / The pancreas is a vital organ that secretes key metabolic hormones and digestive enzymes. In pancreatic ductal adenocarcinoma (PDAC), one of the leading causes of cancer-related death in the world, limited advances in diagnosis or therapies have been made over decades. Key features of PDAC progression include an elevated matrix sti ness and an increased deposition of extracellular matrices (ECM), such as hyaluronic acid (HA). Understanding how cells interact with components in the tumor microenvironment (TME) as PDAC progresses can assist in developing diagnostic tools and therapeutic treatment options. In recent years, hydrogels have proven to be an excellent platform for studying cell-cell and cell-matrix interactions. Utilizing chemically modi ed and naturally derived materials, hydrogel networks can be formed to encompass not only the components, but also the physicochemical properties of the dynamic TME. In this work, a dynamic hydrogel system that integrates multiple click chemistries was developed for tuning matrix physicochemical properties in a manner similar to the temporally increased matrix sti ness and depositions of HA. Subsequently, these dynamic hydrogels were used to investigate how matrix sti ening and increased HA presentation might a ect survival of PDAC cells and their response to chemotherapeutics.

Some have suggested acute increases in musculotendinous stiffness (k) following whole body vibration (WBV). Others propose that chronic ankle instability (CAI) may alter k of the lower extremity. Changes in proprioceptive activity and/or gamma motoneuron activation post-WBV and/or due to CAI could lead to alterations in k. However, little is known about acute effects of WBV on k and less is known about changes in k with CAI. PURPOSE: Assess differences in vertical and joint k between healthy and CAI subjects during single-limb landings and detect alterations in k measures post-vibration. METHODS: Subjects were identified as CAI via the FAAM, MAII and special testing. Thirty-five CAI subjects (17 males, 18 females; age = 22 ± 7 yr; height = 1.73 ± 0.23 m; mass = 70 ± 30 kg) and 35 matched healthy subjects (17 males, 18 females; age = 23 ± 5 yr; height = 1.73 ± 0.21 m; mass = 70 ± 35 kg) qualified for this study. Kinetic (2000 Hz) and kinematic (250 Hz) data were recorded during several jump landings pre- and post-WBV. Five repetitions of WBV, at 26 Hz and 4 mm amplitude, were introduced between pre- and post-WBV jump trials. The jump task included a double-limb jump followed by a single-limb landing and a subsequent contralateral hop. Vertical k (∆vertical GRF/center of mass vertical displacement), hip, knee and ankle joint k (∆joint moment/∆joint angle) were calculated, averaged across five successful pre-WBV and across six post-WBV trials. An ANOVA was used to detect between-group differences, while an ANCOVA was used to analyze within-group differences post-WBV using pre-measures as covariates. A pseudo-Bonferroni adjustment was performed prior to statistical analysis (p < 0.01). RESULTS: No between-group differences were observed for any of the variables (F1,68 = 0.020 to 1.400, p = 0.240 to 0.890). A significant increase in vertical k was observed post-WBV for the healthy group (t67 = 2.760, p = 0.008), but not for the CAI group (t67 = 0.370, p = 0.720). The CAI group did demonstrate a decrease in ankle (t67 = -3.130, p = 0.003) and knee (t67 = -3.490, p = 0.001) joint k post-vibration. No other within-group differences were observed post-WBV (p > 0.01). CONCLUSIONS: It appears that WBV does acutely increase vertical k in healthy subjects. However, this treatment effect was not observed in CAI. Further research is needed to assess how k is regulated in CAI subjects and why CAI subjects responded differently to WBV.

Wrist orthoses are the most common upper limb orthoses, being used by thousands of individuals each year to stabilize, immobilize, or support the wrist joint. Wrist orthoses achieve their function by altering the stiffness of the wrist joint (Figure 1-1). However, there is no quantitative understanding of how wrist orthoses affect wrist stiffness, and consequently, wrist orthosis development often relies on feel, intuition, or empirical heuristics rather than a methodical, quantitative approach. Because wrist movement control is dominated by wrist joint stiffness (Charles and Hogan 2011) a quantitative understanding of how wrist orthoses alter the stiffness of the wrist is imperative to the development of improved wrist orthoses with properties tailorable to the needs of the thousands of individuals who use them. In order to begin bridging this gap, our research characterized the stiffness of four common groups of wrist orthosis in two degrees of freedom: flexion-extension (FE) and radioulnar deviation (RUD) which are the degrees of motion most affected by wrist orthoses. We used a wrist robot to measure how twelve orthoses altered the passive wrist stiffness of twenty healthy subjects (three orthoses and five subjects per orthosis group). To perform these measurements we designed a unique wrist-mounting fixture (Figure 3-2) which allows the wrist robot to manipulate the hand inside an orthosis without interfering with orthosis motion (more accurately simulating the actual hand-orthosis interaction). Our results showed that (1) three out of four orthosis groups significantly altered the stiffness of the wrist joint, (2) orthoses in the same group are not generally significantly different than one another, and (3) there are important differences in stiffness between different orthosis groups. An interesting implication of our research is the result that in many cases orthoses with volar stays may be interchanged with orthoses with both volar and dorsal stays without significant changes in orthosis performance (Table 4-2). We anticipate this work will prove fruitful toward the future study of wrist orthoses' effects on wrist movement behavior and the future improvement of wrist orthosis design.