Bioengineering Seminar Schedule, Spring 2008
"Small Molecule Functionalized Capture Materials for Functionally-Directed Brain Proteomics and Neurotransmitter Sensing"
Amit Vaish, Penn State University
Friday, January 18, 12:00 - 1:00 p.m., Room 210 Hallowell, CG624E Hershey
Abstract
Novel methods have been developed to tether low molecular weight neurotransmitter molecules to highly optimized biospecific self-assembled monolayer (SAM) surfaces. Both serotonin and 5-hydroxytryptophan (5-HTP) functionalized SAMs have been fabricated at low surface coverages using co-deposition of mixed monolayers from solution or insertion of tethers into preformed SAMs at ultralow densities. These materials have been investigated for their ability to specifically recognize large biomolecule binding partners using quartz crystal microbalance (QCM) gravimetry and fluorescence spectroscopy. Both types of materials show low nonspecific binding and serotonin-functionalized SAMs selectively capture anti-serotonin antibodies vs. antibodies directed against other neurotransmitters with similar structures. SAMs functionalized with the serotonin precursor, 5-HTP, capture recombinant receptors that natively recognize free serotonin. In addition, to selectivity, binding kinetics have been investigated using QCM to determine equilibrium binding constants. These surfaces are being used in conjunction with mass spectrometry for the detection, structural identification and association of functionally-related proteome sets and to select for short RNA or DNA oligomers (aptamers) that will be used as high affinity molecular recognition elements for the development of small, fast and highly sensitive in vivo neurotransmitter sensors capable of achieving the spatial and temporal resolution necessary for real-time physiological studies.
"Magnetic Resonance at the Microscale"
Andrew Webb, Penn State University
Friday, January 25, 12:00 - 1:00 p.m., Room 210 Hallowell, CG624E Hershey
Abstract
Conventional clinical magnetic resonance imaging can detect pathologies and structures with dimensions of a few mm or more. In animal studies, the dimensions are reduced to several hundreds of microns. However, by designing specialized radiofrequency and gradient hardware it is possible to obtain images with spatial resolution less than 10 microns. This type of approach has also been applied to high resolution and solid state NMR spectroscopy, enabling improvements in limits-of-detection of more than an order-of-magnitude. This talk will outline the design criteria necessary to achieve high spatial resolution and improved limits-of-detection, and discuss some of the practical applications relevant to single cell studies, pharmaceutical applications, producing models for computational fluid dynamics, and embryology.
NO SEMINAR
Friday, February 1, 12:00 - 1:00 p.m., Room 210 Hallowell, CG624E Hershey
Weather delay.
"Nanoparticle Plasmonics: A Versatile Platform for Molecular Imaging and Bio-Sensing"
Jesse Aaron, University of Texas at Austin
Friday, February 8, 12:00 - 1:00 p.m., Room 210 Hallowell, CG624E Hershey
Abstract
Conventional clinical magnetic resonance imaging can detect pathologies and structures with dimensions of a few mm or more. In animal studies, the dimensions are reduced to several hundreds of microns. However, by designing specialized radiofrequency and gradient hardware it is possible to obtain images with spatial resolution less than 10 microns. This type of approach has also been applied to high resolution and solid state NMR spectroscopy, enabling improvements in limits-of-detection of more than an order-of-magnitude. This talk will outline the design criteria necessary to achieve high spatial resolution and improved limits-of-detection, and discuss some of the practical applications relevant to single cell studies, pharmaceutical applications, producing models for computational fluid dynamics, and embryology.
"Power of Lab-on-a-Chip: from Microvascular Networks to Embedded Batteries"
Sergey Sherkoplas, Harvard Medical School - Massachusetts Institute of Technology
Monday, February 11, 12:00 - 1:00 p.m., Room 210 Hallowell, CG624E Hershey
Abstract
I will describe the development of a microchannel network as a platform for studying the mechanics of blood flow in the microcirculation, and the use of this microcirculation-on-a-chip as a tool for quantifying the effect of deformability of red blood cells on perfusion of microvascular networks. In addition, I will show how the natural, scale-specific properties of blood flow in the microchannel network can be used for positive, passive, continuous-flow separation of white blood cells from whole blood. Finally, the fabrication of an embedded primary battery for powering disposable lab-on-a-chip devices will be discussed.
NO SEMINAR - Research Day in Hershey
Friday, February 15, 12:00 - 1:00 p.m., Room 210 Hallowell, CG624E Hershey
FINAL DEFENSE - "Experimental and Theoretical Investigations of Kinesin-2 Mechanochemistry"
Gayatri Muthukrishnan, Penn State University
Friday, February 22, 12:00 - 1:00 p.m., Room 210 Hallowell, CG624E Hershey
Abstract
Of the 14 kinesin families, Kinesin-2 motors are unique in having two different motor domains. These motors are involved in intraflagellar transport as well as cytoplasmic transport of melanosomes. Mechanistic differences between Kinesin-1 and Kinesin-2 motors may provide insights into their underlying hydrolysis cycles. Optical tweezers with back focal plane interferometry detection system was constructed and calibrated to aid experiments at the single molecule level. This system was then used to manipulate polystyrene beads functionalized with single Kinesin-1 or Kinesin-2 motors, and run lengths and velocities were measured under minimal loads. Mouse KIF3A/B was compared to homodimeric chimeras, KIF3A/A and KIF3B/B and to conventional Kinesin-1 motors. At saturating ATP, KIF3A/B moved at 436 ± 129 nm/s and the homodimers moved at similar speeds, while Kinesin-1 moved at 702 ± 136 nm/s. The run lengths of all three KIF3 motors were approximately 600 nm, while the run length for Kinesin-1 was three-fold higher. When ATP concentrations were reduced from 1 mM down to 1 µM, Kinesin-1 run lengths were constant, consistent with previous reports. This implies that Kinesin-1 waits in the same chemomechanical state regardless of the time it takes for ATP to bind. In contrast, the run length of KIF3A/A increased nearly three-fold when the ATP was lowered from 1 mM ATP to 1 µM. This implies that during the time the motor waits for ATP to bind at limiting ATP levels, the motor transitions to a different chemomechanical state, resulting in a lower probability of detachment following ATP binding. Stochastic simulation of motor stepping showed that at saturating concentrations, ATP binds to the motor when both heads are bound to the microtubule. This result puts constraints on the kinetic cycles for Kinesin-1 and Kinesin-2 motors.
"Functional Effects of the Extracellular Mechanics in Cardiovascular Regenerative Medicine"
Jeffrey Jacot, University of California, San Diego
Friday, February 29, 12:00 - 1:00 p.m., Room 210 Hallowell, CG624E Hershey
Abstract
Successful implementation of regenerative cardiovascular therapies requires the development of technologies
to control spatial alignment and mechanical function of individual cells. Using novel techniques for measuring
macroscale and microscale compliance of bypass grafts during postsurgical remodeling, I characterized remodeling
mechanisms that effectively reduce wall tension. I also developed an in vitro model of arterial injury that could
resolve spatial patterns in proliferation and found that local intercellular signaling leads to focal regions of
high proliferation. In addition, using mechanically tunable substrate systems, I observed that the stiffness of
the extracellular matrix can affect the maturation of cardiac myocytes, resulting in differences in shape, structure,
protein expression and contractile behavior. I have observed similar effects on the differentiation and development
of embryonic stem cells on engineered materials and I will discuss how this could translate to the development
of implantable tissues to treat heart defects and disease.
FINAL DEFENSE - "Mechanisms of Shear-Induced Adaptive Rheology in Endothelial Cells"
Jhanvi Dangari, Penn State University
Friday, March 7, 12:00 - 1:00 pm, Room 210 Hallowell, CG624E Hershey
Abstract
Vascular endothelial cells (ECs) respond to temporal and spatial characteristics of hemodynamic forces by alterations in their adhesiveness to leukocytes, secretion of vasodilators, and permeability to blood-borne constituents. These physiological and patho-physiological changes are tied to adaptation of cell mechanics and mechanotransduction, the process by which cells convert forces to intracellular biochemical signals. While long term shear adaptation of endothelial cells has been studied extensively, little is know about the temporal changes in cell mechanics occurring at short time scales (on the order of seconds). We used the method of particle tracking microrheology to study adaptive changes in intracellular mechanics in response to a step change in fluid shear stress, which simulates both rapid temporal and steady features of hemodynamic forces. Results indicate that endothelial cells become significantly more compliant as early as 30 seconds after step change in shear stress from 0 to 10 dynes/cm2 followed by recovery of viscoelastic parameters within 4 minutes of shearing even though shear stress was maintained. Further, we investigated the role of actomyosin interactions in the dynamic control of EC mechanics. A novel observation from these studies was that actomyosin regulates macrorheology (i.e. viscoelastic deformation in response to shear stress), microrheology (i.e. constrained thermal motion of small beads) and cellular activation. In fact, the observation of a shear-induced, actin-dependent contraction the onset time of which depended on myosin II may point the way to a new understanding of the dynamic control of cell mechanics. Together, these studies provide new insight into how early mechanotransduction events may depend on dynamic modulation of mechanical properties and suggest that dynamic adaptive rheology may be one link between hemodynamic force and vascular disease.
NO SEMINAR - Spring Break
Friday, March 14, 12:00 - 1:00 p.m., Room 210 Hallowell, CG624E Hershey
"Electrophysiological Characterization of Structured and Stem Cell-Derived Cardiomyocyte Monolayers for Tissue Engineering Applications"
Elizabeth Lipke, Johns Hopkins University
Monday, March 17, 2008, 11:00 a.m. - 12:00, 210 Hallowell, CG623 Hershey
Abstract
Heart disease is the number one cause of death for both men and women in the United States each year. By improving our ability to repair the damaged or diseased heart, we can decrease the number of patients for which a transplant is necessary and give those not eligible for a transplant the opportunity for a better quality, and possibly longer, life. To accomplish this goal, we must be able to create engineered cardiac tissue that can functionally integrate with native myocardium without inducing deadly cardiac arrhythmias. Although a number of groups have made very substantial contributions to the area of cardiac tissue engineering, only limited studies have been done to assess the electrophysiological properties of these engineered tissues. I will present our electrophysiological characterization of large-scale (3.5 cm2) cardiac tissue monolayers, including investigating the guidance of engineered cardiac tissue structure by nanopatterned poly(ethylene)glycol hydrogels and assessing the potential of stem cell-derived cardiomyocytes as a cell source for cardiac repair. Although the structure of cardiac tissue is highly organized in vivo, cardiomyocytes lose their native organization and adopt a random orientation when cultured in vitro. We have shown that by creating PEG hydrogels with tightly controlled, nanoscale structure and using these scaffolds as a base for our cardiac tissue monolayers, the macroscale alignment of the resulting tissue monolayers can be directed. This alignment, in turn, alters the functional properties of the resulting tissue, causing it to better mimic the architecture and function of native myocardium. Adult cardiomyocytes can not be readily expanded in culture. Hence, an alternative cell source is needed for cardiac regeneration strategies to become a reality. Embryonic stem cell-derived cardiomyocytes (ESC-CMs) have the potential to supply the large numbers of cells needed and can meet the very distinct electrophysiological requirements. Using mouse ESC-CMs, we have created large-scale, spontaneously contracting tissue monolayers. We found that these ESC-CM tissue monolayers supported cardiac action potential propagation and could be electrically paced up to an average of 6 Hz. In addition to the potential long-term clinical applications, these engineered cardiac tissues could serve as a platform to test various pharmacological effects on cardiac development and electrophysiology.
"Reflex Contributions to the Regulation of Multijoint Mechanics"
Eric Perreult, Northwestern University
Friday, March 21, 12:00 - 1:00 p.m., Room 210 Hallowell, CG624E Hershey
Abstract
Tool use and object manipulation are fundamental to our existence. To interact with the variety of objects we
encounter in a typical day, the human neuromotor system must regulate the mechanical properties of our limbs to
maintain stability in the in the presence of changing and often uncertain loads. Our laboratory is interested in
understanding the neural and biomechanical mechanisms contributing to that regulation. These studies contribute
to our understanding of the unimpaired motor system as well as to the mechanisms underlying motor impairments following
injuries such as stroke and spinal cord injury. They also form the basis of our current approaches to the design
of advanced rehabilitation technologies.
In this presentation, I will cover our recent studies into the stretch-evoked involuntary mechanisms contributing
to the regulation of arm mechanics. There are multiple neural pathways that can contribute to the nervous system's
response to external perturbations of posture. Often these are grouped under the terminology of stretch-sensitive
reflexes. Once thought to be immutable, it is now well understood that stretch reflexes can be modulated in a task-dependent
manner. However, the purpose of this adaptation remains unclear. A common proposal is that stretch reflexes contribute
to the regulation of limb stability, as is essential for tool use and locomotion on slippery surfaces. Alternatively,
prior to movement onset, stretch reflexes can assist an imposed stretch, opposite to what would be expected from
a stabilizing response. Our results demonstrate that these behaviors are mediated through different neural pathways,
partially clarifying why previous attempts to attribute a single functional role to the stretch reflex have proven
elusive. After presenting these results, I will demonstrate how stretch reflexes contributing to limb stability
depend on the mechanical properties of the human limb in relation to that of the physical environment with which
it is interacting.
"Gas Embolotherapy: A Vascular Microbubble Approach for Tumor Treatment"
Joseph Bull, University of Michigan
Friday, March 28, 12:00 - 1:00 p.m., Room 210 Hallowell, CG624E Hershey
Abstract
Embolotherapy involves the occlusion of blood flow to tumors to treat a variety of cancers, including renal carcinoma and hepatocellular carcinoma. The accompanying liver cirrhosis makes the treatment of hepatocellular carcinoma by traditional methods difficult. Previous attempts at embolotherapy have used solid emboli, such as blood clot, gelatin sponge, particulates, balloons and streamers. A major difficulty in embolotherapy is restricting delivery of the emboli to the tumor, i.e. minimizing ischemia of healthy tissue, without extremely invasive procedures. We are developing a novel minimally invasive gas embolotherapy technique that uses gas bubbles rather than solid emboli. The bubbles originate as encapsulated liquid perfluorocarbon droplets that are small enough to pass through capillaries. The droplets can be selectively vaporized in vivo by focused high intensity ultrasound to form gas bubbles which are then sufficiently large to lodge in the tumor vasculature. Understanding the potential bioeffects from acoustic droplet vaporization and the mechanisms of emboli transport and lodging are essential to designing treatment strategies that achieve highly selective delivery of the gas emboli to the tumor. This seminar will describe our recent studies the biofluid dynamics of microbubbles for therapy, which utilize a combination of experimental and theoretical approaches.
FINAL DEFENSE - "Controlled Assembly of Microtubles and Manipulation of Kinesin Driven Microtubule Motion"
Maruti Uppalapati, Penn State University
Monday, March 31, 1100 - 12:00 p.m., Room 210 Hallowell, CG623 Hershey
Abstract
One of the outstanding problems in nanotechnology is the difficulty of manipulating and transporting materials at the micro- and nano-scales. Motor proteins, such as kinesins, are ideal for active transport of materials at this size range. Kinesins are microtubule based motor proteins that play a major role in intracellular transport of cargo and cell division in eukaryotic cells. These motor proteins utilize the chemical energy of ATP hydrolysis to walk on protein filaments, called microtubules. Kinesins and microtubules can be purified and the motor-driven motion can be reconstituted /in vitro/. By incorporating kinesins and microtubules in synthetic environments, the transport abilities of these proteins can be utilized for applications in biological sensing, micro-actuation and nano-scale manipulation of materials. In addition, nanotechnology can be used to develop tools to study the cellular function of kinesins and microtubules. Progress towards these goals will be discussed.
Student Presentations: Florly Ariola, Byeong-Yuel Lee, BuSik Park
Friday, April 4, 12:00 - 1:00 p.m., Room 210 Hallowell, CG624E Hershey
"Lateral Heterogeneity and Diffusion Patterns of Major Histocompatibility Complex (MHC) Class I Proteins in L Cells"
Florly Ariola
Abstract
Major histocompatibility complex (MHC) class I proteins (H2Ld), which are expressed in almost all nucleated cells, play an important immunological role by delivering antigens to T cells. In the ER, where these molecules are constructed and retained, MHC class I proteins are associated with a series of proteins that assist in peptide loading. Upon peptide loading, the peptide-MHC class I complex is released from the ER and transported through the Golgi apparatus to the plasma membrane. Here, we examined the lateral heterogeneity and diffusion patterns of GFP-encoded constructs with MHC class I proteins in mouse fibroblast cells (L cells). In particular, we compared two constructs, H2LdGFPin and H2LdGFPout, which are GFP tagged at intracellular and extracellular segments of the protein, respectively. Key to our studies are integrated biophotonics techniques, on a single platform, which are employed for investigating multiscale (picoseconds to seconds) dynamics of these constructs within segments of the plasma membrane (i.e., blebs) isolated from L cells. The spatial distribution of these MHC class I proteins was monitored before and after treatment with hydrogen peroxide using DIC and confocal microscopy. Further, the fluorescence properties of GFP-tagged constructs are compared with free GFP using fluorescence lifetime measurements. In addition, ultrafast conformational changes and rotational diffusion of H2LdGFPin and H2LdGFPout are investigated using time-resolved fluorescence polarization anisotropy. Lateral diffusion and molecular brightness of these constructs are being quantified, at the single-molecule level, using fluorescence correlation spectroscopy.
"In vivo Study of Restless Legs Syndrome with Magnetic Resonance Spectroscopy: Exploring Iron Deficiency Consequence in RLS"
Byeong-Yuel Lee
Abstract
The native reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in eukaryotic cells are key metabolic cofactors in redox reactions and energy metabolic pathways. Recently, there is resurgence in employing cellular autofluorescence as a natural probe for diagnostic purposes in cancer, diabetes, apoptosis, and neurodegenerative diseases. Here, I will present quantitative, fluorescence-based biochemistry of intrinsic NADH and FAD at the single-cell level. Cancerous (Hs578T) and normal (Hs578Bst) breast cells were used as a model system. This quantitative fluorescence dynamics assay includes two-photon fluorescence lifetime and polarization anisotropy imaging for contrast, concentration measurements, and analysis of the free-to-enzyme-bound molar fraction. Such non-invasive assay on living cells provides an alternative approach for (i) probing mitochondrial anomalies associated with cell pathology (without the need for exogenous dyes) and (ii) avoiding cell destruction required in conventional biochemical techniques (i.e., loss of the morphological context). The sensitivity of this fluorescence dynamics assay has also been examined in primary and Ras-activated keratinocytes as well as MCF-7 breast cancer cells.
"Numerical Model of a Cylindrical Dielectric Resonator for High Field MRI"
BuSik Park
Abstract
Dielectric resonators have been used for integrated microwave filters and oscillators because of their very high Q values of up to several thousand, lower conductor losses, and smaller size than metallic resonant cavities. Capitalizing on these properties, some researchers have used high dielectric materials in MRI and EPR. Based on previous research , we have designed a dielectric resonator for high field microimaging in MRI and performed full Maxwell numerical calculations of the electromagnetic fields to evaluate the resonator. Here the effect of sample diameter on field uniformity is explored.
Student Presentations: Kunal Paralikar, Pranav Soman, Xiaole Mao
Friday, April 11, 12:00 - 1:00 p.m., Room 210 Hallowell, CG624E Hershey
"Automated Reduction of Non-Neuronal Signals from Intracortical Microwire Array Recordings by Use of Correlation Technique"
Kunal Paralikar
Abstract
Implanted intracortical micro-electrode arrays record multi-unit spike activity that is used in deciphering the neural basis for adaptation, learning, plasticity and as command signal for brain-machine interfaces. In studies with awake and behaving animals, micro-electrode arrays typically also record non-neuronal signals induced by an active animal's movement, feeding and grooming actions. The spectral and temporal nature of these artifacts is similar to neural spikes and hence complicates their accurate detection. Here, an objective preprocessing technique is proposed to identify non-neuronal signals. Its efficacy is demonstrated by evaluating changes in mean-spike waveforms generated from traditional detection schemes after preprocessing based on intra-electrode correlation calculation.
"Measuring the Time-Dependent Functional Activity of Adsorbed Fibrinogen by Atomic Force Microscopy"
Pranav Soman
Abstract
Following adsorption to a surface, fibrinogen is believed to undergo conformational changes that transiently expose the platelet binding epitope in the gamma-chain dodecapeptide (400-411). In this work, we detect the time-dependent functional changes in adsorbed fibrinogen by measuring antigen-antibody debonding forces by atomic force microscopy (AFM). AFM probes were functionalized with monoclonal antibodies recognizing fibrinogen gamma 392-411, which includes the platelet binding dodecapeptide region. Data from multiple fibrinogen force distributions was used to calculate the probability of antigen recognition as a function of fibrinogen residence time. Statistical analysis showed that the probability of antibody-antigen recognition peaked at ~45 minutes post-adsorption and decreased with increasing residence time. Platelet adhesion was determined to be highest for fibrinogen residence times of ~45 minutes, which correlates well with the functional activity of adsorbed fibrinogen as measured by a modified AFM probe. This result suggests that the exposure of the dodecapeptide epitope is primarily responsible for platelet adhesion and that platelet adhesion will peak at 45 minutes fibrinogen residence time on hydrophilic mica substrate.
"Tunable Liquid Gradient Refractive Index (L-GRIN) Lens"
Xiaole Mao
Abstract
Student Presentations: Daniel Gilbert, Andrew Lutes, Ning Yang
Friday, April 18, 12:00 - 1:00 p.m., Room 210 Hallowell, CG624E Hershey
"Effects of Stimulation Rate on Loudness Threshold for Objective Cochlear Implant Fitting"
Daniel Gilbert
Abstract
Cochlear implant technology is continually reaching a broader audience as the FDA-approved age for implantation is lowered below 12 months to take advantage of the heightened neural plasticity in early development stages. Active children, as well as those with undeveloped communication skills, present a challenge when attempting to program a comfortable current range for the implant using the standard subjective tests. User-defined loudness thresholds and the onset of the electric stapedial reflex have been demonstrated to occur at strongly correlated current settings, offering a potential objective diagnostic solution. In this study, the stapedius muscle electromyogram (stEMG) was used as an objective measurement tool to evaluate the effects of the increasingly popular high-rate stimulation on loudness perception.
"Autofluorescence Dynamics and Concentration of Intrinsic Flavin at the Single Cell Level"
Andrew Lutes
Abstract
Flavin adenine dinucleotide (FAD) exists as a metabolic cofactor in cells. The identification of a 800-1000 nm spectral window in which intrinsic FAD and other flavins are selectively excited has empowered scientists to study these biomolecules in living, intact cells of varying pathological states and answer questions about the molecular dynamics and concentration of cellular flavins that have not been satisfactorily addressed. Here, we use several modalities of two-photon (2P) fluorescence microscopy and spectroscopy at 900 nm to reveal new insights about the excited-state lifetime and rotational dynamics of flavins in normal (HTB 125) and breast cancer (HTB 126) cells. Fluorescence lifetime imaging microscopy (FLIM) reveals that cancer cells exhibit significantly faster average decays (biexponential, tfl=1.33±0.08 ns, n=5) than their non-transformed counterpart (biexponential, tfl=1.56±0.08 ns, n=7), and this result is corroborated using single-point magic angle measurements. Using a newly developed image processing algorithm, 2P-FLIM images were converted to concentration maps, which exposed differing flavin concentrations in normal (173±84 µM) and cancer (21±10 µM) cells. In addition, steady-state and time-resolved fluorescence polarization anisotropy measurements indicate a restricted environment. For comparison, the 2P-lifetime measurements of free FAD and bound to LipDH demonstrate an enhanced lifetime for the bound conformation. The results of this study shed new light on the molecular dynamics and protein environment in differing cell pathologies and could facilitate the development of innovative, minimally invasive diagnostic tools for early detection of breast cancer in patients, which could complement or replace existing methods.
"Numerical Study of Blood Flow at the End-to-Side Anastomosis of a Left Ventricular Assist Device for Adult Patients"
Ning Yang
Abstract
Ventricular assist devices (VADs) have been used for years in adult patients with end-stage heart failure, during
bridge-to-transplant, and they have recently shown promise in aiding in myocardial recovery. An area which must
be more adequately addressed is the outflow cannula attached as an end-to-side anastomosis to the aorta. This attachment
may lead to unnaturally high and low shear stresses, turbulence, flow separation, and stagnant flows. As a result,
platelet activation and thrombus formation may occur.
This study was undertaken to advance the understanding of the turbulent blood flow within a rigid engineering model
of the cannulated aorta under continuous flow conditions. Time-accurate Navier-Stokes computational fluid dynamics
(CFD) was used to simulate the flow. An implicit large-eddy simulation (ILES) approach was used which resolves
much of the turbulence spectra and models the length scales below an implicit grid cut-off. A resistance boundary
condition was developed to properly model the resistance of the secondary vessels on the aortic arch. A code-to-code
comparison was performed in a human carotid model under a steady flow condition to verify the CFD code. A systemic
grid study was performed to study the effect of grid size on mean flow field and resolved energy spectra.
The numerical results showed that the proximal configuration caused a higher exit jet flow in the aorta than did
the distal configuration. Turbulent flow was observed near the cannulation site for both proximal and distal anastomotic
configurations. Stagnant flow was found near the aortic root for the distal configuration when the natural heart
was not functioning. Although the proximal configuration effectively reduced the stagnant flow near the aortic
root observed within the distal configuration, it directed a high-velocity jet into the brachiocephalic artery
and left common carotid artery, which might increase the perioperative stroke rate. A large increase (up to 70%)
in flow rate to the secondary vessels was found for the cannulated aortic models when compared to the pre-anastomotic
aorta. The distal configuration caused higher flow rates at the secondary branches than did the proximal configuration.
High wall shear regions (up to 2321 s-1) were found on the aortic arch near the cannulation sites. The proximal
configuration caused a higher wall shear rate distribution when compared to the distal configuration. In general,
the numerical results suggest that the outflow cannulae induce dangerous flow structures in the adult aorta due
to high exit jet flows. This work builds a foundation for future physiologically realistic simulation under pulsatile
flow conditions.
"Analysis of Cells in Blood using BioMEMS"
Siyang Zheng, California Institute of Technology
Monday, April 21, 11:00 - 12:00 p.m., Room 210 Hallowell, CG 624E
Abstract
Blood analysis holds the promise of a revolution in disease diagnosis and therapeutic monitoring. Point-of-care
(POC) has the potential to significantly change healthcare delivery and improve patient outcomes. In the first
half of my talk, I will present bioMEMS enabled systems, handheld blood counters, for normal blood cells analysis
as a POC application. Red blood cell (RBC) and white blood cell (WBC) counts are achieved with electrical impedance
sensing. Improvements on electrode material and sensing mechanism greatly improve the system sensitivity. In another
device, hydrodynamic separation RBC and WBC by size with high purity is demonstrated. Finally, high through-put
WBC differential count (lymphocytes vs. granulocytes) from undiluted blood is achieved with fluorescent optical
detection using a handheld system prototype.
In the second half of my talk, an example of bioMEMS devices for extremely rare blood cells analysis, circulating
cancer cell (CTC) enrichment from blood for early cancer diagnosis and treatment monitoring, will be introduced.
The current CTC capture and identification has significant barriers including the rareness of the CTC (1 out of
10 billion blood cells), multiple procedural steps, handling of relatively large volumes of blood, substantial
human intervention, extremely high cost, and the lack of reliability for the detection methods. I will introduce
the development of a novel micro filtration technology with 90% capture efficiency and 107 enrichment. Testing
results from the model system and clinical results from cancer patient samples show significantly improvements
of this technology over the only FDA approved commercial system. Pathological analysis by immunohistochemical staining,
genetic analysis by cell electrolysis and PCR/RT-PCR, genomic analysis by laser microdissection and comparative
genomic hybridization, can be performed directly on the devices. The more recent 3D microfiltration devices are
capable of capturing viable CTCs and culture them for at least two weeks. The novel microdevice based CTC enrichment
technology will provide a cost effective method for CTC monitoring with higher capture efficiency, higher enrichment,
faster processing time and more reliable results, which can benefit both the cancer clinic and basic cancer research.
"The Push and Pull of Chemical and Mechanical Forces on DNA"
Brian Todd, NIH
Wednesday, April 23, 11:00 - 12:00 p.m., Room 210 Hallowell, CG623
Abstract
With meters of DNA crammed into each microscopic nuclease, the intermolecular forces involved in packing and
unpacking a genome are important regulators of gene expression. We directly measured DNA packing forces in a simplified
genome at the single-molecule level. Our magnetic tweezers measurements allow us to delineate the biologically
relevant roles of osmotic pressure, cation composition, and mechanics on DNA packing. At equilibrium, all of these
influences tie together beautifully via a Gibbs-Duhem equation. Analysis of the electrolyte dependence of these
forces helps us choose an effective polyelectrolyte model for DNA and illuminates the elusive mechanism for counterion-induced
attractions between like-charged objects. Future work leans toward understanding the role of chromatin packing
forces in eukaryotic (e.g. human) gene regulation.
Student Presentations: Lujia Gao, Brett Weaver, Kathryn Richards
Friday, April 25, 12:00 - 1:00 p.m., Room 210 Hallowell, CG624E Hershey
"Measurement of the Permeability of the Endothelial Glycocalyx Using Indicator Dilution Techniques"
Lujia Gao
Abstract
A New method is presented to quantify changes in the permeability of the endothelial glycocalyx to solutes and fluid flow using techniques of indicator dilution. The endothelial cell surface glycocalyx is a proteoglycan based carbohydrate-rich layer, functioning as a barrier to cell adhesion and transvascular exchanges of water and solutes and serving as a depository for receptors of the thrombotic processes, intercellular signaling and more. Several studies have observed that during pathological conditions, the endothelial glycocalyx may be degraded, as for example, in inflammation, diabetes and hypercholesterolemia. The present study used a combination of in vivo experiments and computer simulations to quantitatively measure the barrier function to transvascular exchange without disturbing its structure and functions. Using the indicator dilution technique, a bolus of fluorescent solute FITC was introduced into the rat mesenteric circulation. The transient dispersion of the bolus through post-capillary venule is recorded and analyzed offline. To represent dispersion of the solute as a function of radial position in a microvessel, virtual transit time (VTT) was calculated from the first moment of intensity-time curves. The computer simulation and subsequent in vivo measurements showed that the radial gradient of VTT over the glycocalyx layer (IP-EC DVTT/Dr) positively correlates with hydraulic hindrance in the layer along the axial direction. Following an artificial induced local inflammation by 10-7 M fMLP-Ringer solution, the IP-EC DVTT/Dr exhibits a significant decrease from 0.229±0.082 sec/ m to 0.183±0.086 sec/ m. From computer simulations, the IP-EC DVTT/Dr was found to be governed by three independent variables: the thickness (d), the hydraulic resistivity of the glycocalyx (Kr) and the effective diffusion coefficient of the solute (Deff). Thus changes of the endothelial glycocalyx were mapped to changes in d, Kr and Deff. Results of the present study provide a technique for assessing alteration of the native endothelial glycocalyx and delineating the shedding process and its anisotropic structure.
Brett Weaver
Abstract
"Platelet Activation Levels in Animal Implant Studies"
Kathryn Richards
Abstract
Platelets can become activated in vivo by traumatic events such as increased sheer rates or contact with synthetic material surfaces. This events can trigger platelet activation and may lead to platelet aggregation and thrombosis. In this study we assess platelet activation levels in a sham surgery as a background for use in assessing platelet response to ventricular assist devices. Upon platelet activation, CD63 is translocated to the surface of platelets. A monoclonal antibody against CD63 was used to label activated platelets while a polyclonal antibody against GPIIbIIIa (the platelet integrin receptor) was used to label all platelets. Flow cytometry was used to determine platelet activation levels following surgery by calculating the activation % as the number of platelets expressing CD63 divided by the total number of platelets. Results showed a substantial increase in platelet activation following the sham surgery, with a return to baseline activation levels 1.5-3 weeks after surgery. Furthermore, other stress events were also found to lead to increases in platelet activation. These results offer understanding into the dynamic hematological environment and are critical in assessing response to implanted devices.
"Microfabricated Platforms for Stem Cell Research"
Bonggeun Chung, Harvard-MIT Division of Heath Sciences and Technology
Monday, April 28, 11:00 - 12:00 p.m., 210 Hallowell, CG623 Hershey
Abstract
This paper describes the development and characterization of microfluidic platforms to study proliferation, differentiation, migration, and apoptosis of neural stem cells (NSCs). NSCs hold tremendous promise for fundamental biological studies and cell-based therapies in human disorders. NSCs are defined as cells that can self-renew yet maintain the ability to generate the three principal cell types of the central nervous system such as neurons, astrocytes, and oligodendrocytes. Despite their promise, cell-based therapies are limited by the inability to precisely control their behavior in culture. Compared to traditional culture tools, microfluidic platforms can provide much greater control over cell microenvironments and optimize proliferation and differentiation conditions of cells exposed to combinatorial mixtures of growth factors. NSCs proliferated and differentiated in a graded and proportional fashion that varied directly with growth factor concentration. Directed embryonic stem (ES) cell differentiation is also a potentially powerful approach for generating a renewable source of cells for regenerative medicine. Typical in vitro ES cell differentiation protocols involve the formation of ES cell aggregate intermediates called embryoid bodies (EBs). Recently, we demonstrated the use of poly(ethylene glycol) (PEG) microwells as templates for directing the formation of these aggregates, offering control over parameters such as size, shape, and homogeneity. Despite these promising results, the previously developed technology was limited as it was difficult to reproducibly obtain cultures of homogeneous EBs with high efficiency and retrievability. In this study, we improve the platform by optimizing a number of features: material composition of the microwells, cell seeding procedures, and aggregate retrieval methods. Adopting these modifications, we demonstrate an improved degree of homogeneity of the resulting aggregate populations and establish a robust protocol for eliciting high EB formation efficiencies. Therefore, the development of microfluidic platforms and hydrogel microwell arrays will help in advancing our understanding of brain development and provide a versatile tool with basic and applied studies in stem cell biology.
"Biomedical Applications of Quantitative Microscopy Techniques"
Michelle Dawson, Massachesetts General Hospital
Wednesday, April 30, 11:00 - 12:00 p.m., Room 210 Hallowell, CG623 Hershey
Abstract
The viscoelasticity of cystic fibrotic (CF) mucus greatly reduces the diffusion rate of colloidal particles,
which limits the effectiveness of gene and drug delivery to epithelial cells in the lungs. To develop a better
understanding of the relationship between the mechanical properties of CF sputum and the transport rates of nanoparticle
gene carriers, the rheological properties of CF sputum were correlated with the transport rates of 100-500 nm particles,
which are the approximate size of the majority of gene carriers. Using high resolution multiple particle tracking
to quantify the transport rates of hundreds of individual model gene carriers, we discovered that particle transport
in CF sputum is highly heterogeneous with a small fraction of particles that move many-fold faster than the average
("outliers") (Dawson, JBC, 2003). These outliers are important since only a small fraction of inhaled
particles may need to cross mucus to treat CF.
Multiple particle tracking can be used to determine the mechanical properties of complex fluids with the accuracy
of traditional bulk-fluid rheological techniques, such as strain-controlled cone and plate rheometry (Dawson, submitted
to AJRCCM). Furthermore, multiple particle tracking can be used to characterize the local mechanical properties
of complex fluids within microscopic domains, which are overlooked by bulk-fluid rheological techniques. This technique,
which is often referred to as microrheology, has been used to map the viscoelasticity of the cell cytoplasm (Tseng,
Biophys J, 2002) and to measure the viscoelasticity of concentrated solutions of DNA (Apgar, Biophys J, 2000).
In future studies, I will utilize multiple particle tracking microrheology to characterize the rheological properties
of undifferentiated bone marrow-derived stem cells.
In conditions of increased cell turnover, such as wound healing, tissue remodeling, or bone growth, stem and progenitor cells are recruited from the bone marrow and contribute to the formation of new tissues. Similarly, cellular regeneration is increased in the formation of the tumor stromal compartment. To facilitate this process, tumors express soluble growth factors, including vascular endothelial growth factor (VEGF), angiopoetins, and placental growth factor (PlGF), that stimulate the migration of bone marrow derived cells (BMDCs) to the tumor. Recently, the role of VEGFR (receptor)-1 emerged as a mediator of angiogenesis and metastasis via recruitment of hematopoietic precursor cells to solid tumors and pre-metastatic niches (Nature, 2005). Using confocal microscopy and image analysis software, we quantified the effects of antibodies that block VEGFR1 and its ligand, PlGF, on the recruitment and accumulation of BMDCs in the primary tumor and lungs of immunocompetent mice (first irradiated and rescued by bone marrow transplant from transgenic mice constitutively expressing green fluorescent protein). We found that antibodies directed at VEGFR1 and PlGF, reduced BMDC accumulation; however, only PlGF-blockade reduced tumor growth and metastasis. This study provides quantitative analysis of the kinetics of bone marrow cell accumulation in primary and metastatic tumors.
"Biomechanics of Coronary Circulation"
Ghassan S. Kassab, Purdue University
Friday, May 2, 12:00 - 1:00 p.m., Room 210 Hallowell, CG624E Hershey
Abstract
Our research interests encompass the biomechanics of the coronary circulation in health and disease. One of our goals has been to develop a computational hemodynamic model of the entire coronary circulation based on a detailed set of anatomical and rheological data of the coronary blood vessels. We have previously obtained the necessary set of anatomical and rheological data of the coronary vasculature which serve as the foundation for the large scale analysis. We are also studying the remodeling of the coronary circulation in disease such as hypertrophy and heart failure. Specifically, we are focusing on the structural and mechanical remodeling of the coronary blood vessels in response to physical factors (i.e., principal and shear stresses). The experimental aspects of our investigations encompass the collection of morphological and mechanical data on the coronary blood vessels as well as measurements of the hemodynamic parameters of interest. The theoretical aspects focus on expressing the remodeling data mathematically in order to formulate and solve significant boundary-value problems of the heart.
FINAL DEFENSE - "Development of a Computational Fluid Dynamics Tool to Explore the Interactions Between Cancer Cells and Leukocytes"
Meghan Hoskins, Penn State University
Monday, May 5, 2:30-3:30 p.m., Room 210 Hallowell, CG624E Hershey
Abstract
A new multidisciplinary computational method has been developed to model the physicsof interacting systems of
blood and cancer cells. The method is applied to explore the mechanics of melanoma cell interactions with white
blood cells in a shear flow. The computational technique combines author-developed and commercial software to model
four component physics: fluid dynamics, structural mechanics, six-degree-of-freedom motion, and adhesion biochemistry.
The specific application of the model development is the interactions of melanoma cells with polymorphonuclear
white blood cells (neutrophils, PMNs). It has been shown that human PMNs may link melanoma cells to blood vessel
walls during the metastasis process. To model these interactions, the adhesion parameters specific to melanoma
cell adhesion to PMNs were determined experimentally. Several computational studies were completed to verify and
validate the model components, and the model was used to explore PMN deformation and melanoma cell-PMN collisions
under differing flow conditions.
"Molecular Imaging & Sensing Using Self-illuminating Quantum Dots"
Yun Xing, Stanford University
Monday, May 12, 11:00 a.m. -12:00 p.m., Room 210 Hallowell, CG623 Hershey
Abstract
Semiconductor quantum dots, due to their superior fluorescent properties, have captivated tremendous attention from researchers in the biomedical field during the past decade. However, the utility of existing quantum dots for in vivo imaging is limited because they require excitation from external illumination sources to fluoresce, which results in a strong autofluorescence background and presents a challenge for deep tissue imaging since only a paucity of excitation light is available at these locations due to tissue absorbance and scattering. Recently, our lab has developed the self-illuminating QDs, which completely eliminate the need of external light for excitation and allows deeper and more sensitive imaging of living tissues. The self-illuminating mechanism relies on bioluminescence resonance energy transfer between a bioluminescence protein (donor) and semiconductor QDs (acceptor). Compared with existing quantum dots, self-illuminating quantum dot conjugates have greatly enhanced sensitivity in small animal imaging, with an in vivo signal-to-background ratio of > 1000 for 5 pmol of conjugates. By inserting a protease-specific substrate between the QD and luciferase, the self-illuminating QD can be turned "down/off" in the presence of active enzymes such as MMPs. Using this system, we were able to detect as low as 5 ng/ml of MMP-7 in buffer and serum. Simultaneous detection of multiple proteases in one solution using QD-BRET conjugates has also been achieved. Preliminary animal imaging results demonstrated distinct responses of these probes to tumor versus normal regions inside living mice.
For additional information, contact Ms. Doretta Garvey, Dept of Bioengineering, Tel: 814.865.1407 or E-Mail: bioe@engr.psu.edu