Analysis of Integrin-mediated Cell Adhesion Strengthening Using Surfaces Engineered to Control Cell Shape and Focal Adhesion Assembly

Cell adhesion to extracellular matrix proteins is critical to physiological and pathological processes as well as biomedical and biotechnological applications. Cell adhesion is a highly regulated process involving initial receptor-ligand binding, and subsequent clustering of these receptors and rapid association with the actin cytoskeleton as focal adhesions are assembled. Focal adhesions enhance adhesion, functioning as structural links between the cytoskeleton and the extracellular matrix and triggering signaling pathways that direct cell function. The objective of this thesis research is to develop a mechanical and biochemical analysis of the adhesion strengthening response. Our central hypothesis was that focal adhesion size and position regulate cell adhesion strength by controlling the distribution of mechanical loading. We engineered micropatterned surfaces to control the size and position of focal adhesions in order to analyze the contributions of these specialized adhesive structures to adhesion strengthening. By applying surface micropatterning techniques, we showed robust control over cell-substrate contact area and focal adhesion assembly. Using a hydrodynamic shear assay to quantify adhesion strength to micropatterned substrates, we observed significant adhesive area- and time-dependent increases in adhesion strength. Complimentary biochemical assays allowed us to probe the role of structural proteins recruited to focal adhesions and examine the structure-function relationships between these adhesive structures and adhesion strength. These findings provide insights into the role of focal adhesions in adhesion strengthening, and may contribute to tissue engineering and biomaterials applications.Ph.D.Committee Chair: Garcia, Andres; Committee Member: King, William; Committee Member: LaPlaca, Michelle; Committee Member: Radhakrishna, Harish; Committee Member: Zhu, Chen

Sheathless Size-Based Acoustic Particle Separation

Particle separation is of great interest in many biological and biomedical applications. Flow-based methods have been used to sort particles and cells. However, the main challenge with flow based particle separation systems is the need for a sheath flow for successful operation. Existence of the sheath liquid dilutes the analyte, necessitates precise flow control between sample and sheath flow, requires a complicated design to create sheath flow and separation efficiency depends on the sheath liquid composition. In this paper, we present a microfluidic platform for sheathless particle separation using standing surface acoustic waves. In this platform, particles are first lined up at the center of the channel without introducing any external sheath flow. The particles are then entered into the second stage where particles are driven towards the off-center pressure nodes for size based separation. The larger particles are exposed to more lateral displacement in the channel due to the acoustic force differences. Consequently, different-size particles are separated into multiple collection outlets. The prominent feature of the present microfluidic platform is that the device does not require the use of the sheath flow for positioning and aligning of particles. Instead, the sheathless flow focusing and separation are integrated within a single microfluidic device and accomplished simultaneously. In this paper, we demonstrated two different particle size-resolution separations; (1) 3 µm and 10 µm and (2) 3 µm and 5 µm. Also, the effects of the input power, the flow rate, and particle concentration on the separation efficiency were investigated. These technologies have potential to impact broadly various areas including the essential microfluidic components for lab-on-a-chip system and integrated biological and biomedical applications.Bankhead-Coley Florida Cancer Research Program (Grant # 1BN04-34183)National Science Foundation (U.S.) (Grant 0968736)National Science Foundation (U.S.) (Grant 1135419)National Science Foundation (U.S.) (Grant 1056475

Renal Safety of a Tenofovir-Containing First Line Regimen: Experience from an Antiretroviral Cohort in Rural Lesotho

Current guidelines contraindicate TDF use when creatinine clearance (CrCl) falls below 50 ml/min. We report prevalence of abnormal renal function at baseline and factors associated with abnormal renal function from a community cohort in Lesotho

Insulin Resistance and the IGF-I-Cortical Bone Relationship in Children Ages 9-13 Years

IGF-I is a pivotal hormone in pediatric musculoskeletal development. Although recent data suggest that the role of IGF-I in total body lean mass and total body bone mass accrual may be compromised in children with insulin resistance, cortical bone geometric outcomes have not been studied in this context. Therefore, we explored the influence of insulin resistance on the relationship between IGF-I and cortical bone in children. A secondary aim was to examine the influence of insulin resistance on the lean mass-dependent relationship between IGF-I and cortical bone. Children were otherwise healthy, early adolescent black and white boys and girls (ages 9 to 13 years) and were classified as having high (n = 147) or normal (n = 168) insulin resistance based on the homeostasis model assessment of insulin resistance (HOMA-IR). Cortical bone at the tibia diaphysis (66% site) and total body fat-free soft tissue mass (FFST) were measured by peripheral quantitative computed tomography (pQCT) and dual-energy X-ray absorptiometry (DXA), respectively. IGF-I, insulin, and glucose were measured in fasting sera and HOMA-IR was calculated. Children with high HOMA-IR had greater unadjusted IGF-I (p < 0.001). HOMA-IR was a negative predictor of cortical bone mineral content, cortical bone area (Ct.Ar), and polar strength strain index (pSSI; all p ≤ 0.01) after adjusting for race, sex, age, maturation, fat mass, and FFST. IGF-I was a positive predictor of most musculoskeletal endpoints (all p < 0.05) after adjusting for race, sex, age, and maturation. However, these relationships were moderated by HOMA-IR (pInteraction < 0.05). FFST positively correlated with most cortical bone outcomes (all p < 0.05). Path analyses demonstrated a positive relationship between IGF-I and Ct.Ar via FFST in the total cohort (βIndirect Effect = 0.321, p < 0.001). However, this relationship was moderated in the children with high (βIndirect Effect = 0.200, p < 0.001) versus normal (βIndirect Effect = 0.408, p < 0.001) HOMA-IR. These data implicate insulin resistance as a potential suppressor of IGF-I-dependent cortical bone development, though prospective studies are needed

Anion-Sensitive Regions of L-Type CaV1.2 Calcium Channels Expressed in HEK293 Cells

L-type calcium currents (ICa) are influenced by changes in extracellular chloride, but sites of anion effects have not been identified. Our experiments showed that CaV1.2 currents expressed in HEK293 cells are strongly inhibited by replacing extracellular chloride with gluconate or perchlorate. Variance-mean analysis of ICa and cell-attached patch single channel recordings indicate that gluconate-induced inhibition is due to intracellular anion effects on Ca2+ channel open probability, not conductance. Inhibition of CaV1.2 currents produced by replacing chloride with gluconate was reduced from ∼75%–80% to ∼50% by omitting β subunits but unaffected by omitting α2δ subunits. Similarly, gluconate inhibition was reduced to ∼50% by deleting an α1 subunit N-terminal region of 15 residues critical for β subunit interactions regulating open probability. Omitting β subunits with this mutant α1 subunit did not further diminish inhibition. Gluconate inhibition was unchanged with expression of different β subunits. Truncating the C terminus at AA1665 reduced gluconate inhibition from ∼75%–80% to ∼50% whereas truncating it at AA1700 had no effect. Neutralizing arginines at AA1696 and 1697 by replacement with glutamines reduced gluconate inhibition to ∼60% indicating these residues are particularly important for anion effects. Expressing CaV1.2 channels that lacked both N and C termini reduced gluconate inhibition to ∼25% consistent with additive interactions between the two tail regions. Our results suggest that modest changes in intracellular anion concentration can produce significant effects on CaV1.2 currents mediated by changes in channel open probability involving β subunit interactions with the N terminus and a short C terminal region

Computational Identification of Transcriptional Regulators in Human Endotoxemia

One of the great challenges in the post-genomic era is to decipher the underlying principles governing the dynamics of biological responses. As modulating gene expression levels is among the key regulatory responses of an organism to changes in its environment, identifying biologically relevant transcriptional regulators and their putative regulatory interactions with target genes is an essential step towards studying the complex dynamics of transcriptional regulation. We present an analysis that integrates various computational and biological aspects to explore the transcriptional regulation of systemic inflammatory responses through a human endotoxemia model. Given a high-dimensional transcriptional profiling dataset from human blood leukocytes, an elementary set of temporal dynamic responses which capture the essence of a pro-inflammatory phase, a counter-regulatory response and a dysregulation in leukocyte bioenergetics has been extracted. Upon identification of these expression patterns, fourteen inflammation-specific gene batteries that represent groups of hypothetically ‘coregulated’ genes are proposed. Subsequently, statistically significant cis-regulatory modules (CRMs) are identified and decomposed into a list of critical transcription factors (34) that are validated largely on primary literature. Finally, our analysis further allows for the construction of a dynamic representation of the temporal transcriptional regulatory program across the host, deciphering possible combinatorial interactions among factors under which they might be active. Although much remains to be explored, this study has computationally identified key transcription factors and proposed a putative time-dependent transcriptional regulatory program associated with critical transcriptional inflammatory responses. These results provide a solid foundation for future investigations to elucidate the underlying transcriptional regulatory mechanisms under the host inflammatory response. Also, the assumption that coexpressed genes that are functionally relevant are more likely to share some common transcriptional regulatory mechanism seems to be promising, making the proposed framework become essential in unravelling context-specific transcriptional regulatory interactions underlying diverse mammalian biological processes

Correction to: Cluster identification, selection, and description in Cluster randomized crossover trials: the PREP-IT trials

An amendment to this paper has been published and can be accessed via the original article

Nanoindentation Study of Polydimethylsiloxane Elastic Modulus Using Berkovich and Flat Punch Tips

This article explores polydimethylsiloxane (PDMS) mechanical properties, and presents nanoindentation experiments with Berkovich and flat punch indenters. In the Berkovich tip quasi-static nanoindentation test, there are pull-in and pull-off events observed during the initial tip contact, and when withdrawing from the surface, respectively. The pull-in interaction needs to be accounted for to properly determine the initial contact point, and thus the accurate contact area. Once accounted for the pull-in event, the Berkovich and flat punch tips quasi-static nanoindentation tests give comparable results of about 1.5 MPa for the PDMS elastic modulus (5 : 1 elastomer base to the curing agent ratio). However, PDMS unloading stiffness is higher than the loading stiffness, and dynamic PDMS testing yields higher elastic modulus of about 3.6 MPa. While these results are comparable with the large strain macroscopic compression test results, the difference underscores the complexity of elastomer mechanical characterization and illustrates the discrepancies typical of the reported values. This article describes nanoindentation methods and critical aspects of interpreting results to assess PDMS mechanical properties. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 41384

Crosslinking Effect on Polydimethylsiloxane Elastic Modulus Measured by Custom-built Compression Instrument

A macroscopic compression test utilizing a simple custom-built instrument was employed to measure polydimethylsiloxane (PDMS) elastic modulus. PDMS samples with varying crosslinking density were prepared with the elastomer base to the curing agent ratio ranging from 5 : 1 to 33 : 1. The PDMS network elastic modulus varied linearly with the amount of crosslinker, ranging from 0.57 MPa to 3.7 MPa for the samples tested. PDMS elastic modulus in MPa can be expressed as 20 MPa/PDMS base to curing agent ratio. This article describes a simple method for measuring elastic properties of soft polymeric materials. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 41050

Cell Adhesion Strengthening: Contributions of Adhesive Area, Integrin Binding, and Focal Adhesion Assembly

Mechanical interactions between a cell and its environment regulate migration, contractility, gene expression, and cell fate. We integrated micropatterned substrates to engineer adhesive area and a hydrodynamic assay to analyze fibroblast adhesion strengthening on fibronectin. Independently of cell spreading, integrin binding and focal adhesion assembly resulted in rapid sevenfold increases in adhesion strength to steady-state levels. Adhesive area strongly modulated adhesion strength, integrin binding, and vinculin and talin recruitment, exhibiting linear increases for small areas. However, above a threshold area, adhesion strength and focal adhesion assembly reached a saturation limit, whereas integrin binding transitioned from a uniform distribution to discrete complexes. Adhesion strength exhibited exponential increases with bound integrin numbers as well as vinculin and talin recruitment, and the relationship between adhesion strength and these biochemical events was accurately described by a simple mechanical model. Furthermore, adhesion strength was regulated by the position of an adhesive patch, comprised of bound integrins and cytoskeletal elements, which generated a constant 200-nN adhesive force. Unexpectedly, focal adhesion assembly, in particular vinculin recruitment, contributed only 30% of the adhesion strength. This work elucidates the roles of adhesive complex size and position in the generation of cell-extracellular matrix forces