Yas Oloumi Yazdi
Doctor of Philosophy in Biomedical Engineering (PhD)
Research Topic
Alignment of magnetic microstructures in an injectable spinal cord injury treatment: Simulation and generation of configurable magnetic fields
Karen Cheung
Professor
Research Classification
Research Interests
additive manufacturing
BioMEMS
Biomedical Technologies
biosensors
microfluidics
neural interfaces
organ-on-chip
Tissue Engineering
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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Development of a microfluidic human airway-on-a-chip with cell-laden hydrogel for studying aerosol inhalation exposure with application demonstrated using whole wood smoke (2024)
Over the years climate change has led to an increase in the occurrence and frequency of forest fires worldwide. As these events create smoke containing harmful particulates and gases that are difficult to avoid and can travel long distances, they can have a tremendous impact on public health. For those with pre-existing respiratory diseases the risks associated with exposure are heightened as exposure can exacerbate symptoms resulting in hospitalization and disease progression. To advance our understanding of smoke related risks to respiratory health, there is a need for more physiologically relevant in vitro models, especially those compatible with realistic aerosol-based exposures. Current microfluidic lung-on-a-chip technologies have emerged as promising tools for exposures, however their lack of a realistic 3D ECM and/or vasculature limits their ability to capture cell-cell and cell-ECM interactions. The work in this dissertation focuses on the development of a tri-culture microfluidic in vitro model of the human small conducting airways compatible with aerosolized wood smoke exposure. The airway-on-a-chip leverages a compartmentalized microchannel design in combination with a lumen patterned, cell-laden hydrogel to mimic the airways. The combination of photopolymerizable hydrogel and sacrificial molding enables the device to replicate the airway’s 3D ECM and microarchitecture including the microvasculature which is achieved by using the lumen structure as a vessel template. The hydrogel selection process in which potential hydrogels were assessed by comparing their properties to the native lung ECM is described within this dissertation. Integration of the hydrogel into the microdevice was validated by ensuring that the cells that make up the airway-on-a-chip were healthy and viable, as well were able to recapitulate key biological responses. A controllable dynamic microenvironment and recapitulation of physiological flows and flow-related shear stresses was achieved via two pump-based flow systems. Novel wood smoke exposures performed on the airway-on-a-chip demonstrated the microdevices ability to mimic in vivo-like exposure and recapitulate wood smoke-induced cell damage and inflammation. The dissertation also demonstrates that using a biodegradable film to separate vertically stacked microchannels rather than a traditional synthetic membrane enables direct epithelial-ECM interactions and creates a model free of non-physiological materials at cell-cell and cell-ECM interfaces.
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Improving data acquisition from 3-D microtissues in image-based cancer drug screening (2022)
Cell-based drug screenings are one of the early steps in the cancer drug development pipeline. New drug candidates are tested on cells related to the intended tissues to gather information about the drug efficacy, toxicity, and metabolism, in order to screen the promising drug candidates before animal and human studies. Traditionally, these screenings have been performed using tissue culture plates that grow cells on a two-dimensional surface. However, traditional drug screening methods have a high failure rate in subsequent clinical steps due to their failure to simulate the natural microenvironment of cancer cells. Three-dimensional cultures such as cancer spheroids try to address this problem by growing the cells in 3-D to mimic the in vivo conditions better, simulating the conditions such as low oxygen levels and insufficient nutrients, and including cell-cell interactions that are absent in 2-D cultures. Benefitting from 3-D cultures, while meeting the high-throughput demands of modern industrial practice requires reliance on scalable image-based techniques and developments of applicable tools for this purpose.This thesis showcases several studies using an image-based drug screening microfluidic platform capable of controlling the oxygen concentration around the cells, employing breast cancer cell spheroids. We show that spheroids can grow on-chip while monitoring them with two-photon microscopy. We have studied the effects of hypoxia on lipid nanoparticle absorption in breast cancer tumour spheroids, as well as the effects of hypoxia on cancer spheroids under chemotherapeutic treatment.This thesis also prominently presents HyClear, a novel tissue clearing protocol developed specifically for the rapid and easy application on spheroids and other microtissues, in order to enable imaging them through their depth in a high-throughput manner. HyClear was developed by adapting other tissue clearing protocols (established for larger tissues) to the needs of spheroids. The HyClear protocol was compared with other prominent tissue clearing protocols, to showcase several examples of its ability to clear small and large tissues and its applicability to large screening experiments.
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Improving the performance of silicon photonic optical resonator-based sensors for biomedical applications (2020)
Silicon photonic biosensors show great potential for applications in medical diagnostics and healthcare services. Near-infrared transparency and high refractive index of silicon allow us to build compact and efficient circuits leveraging CMOS foundries, which provide low-cost mass production and enable the integration of the optoelectronic components on the same chip. Although silicon photonic biosensors have proven performances close to today's gold standard diagnostics, many applications still require higher multiplexing, as well as more sensitive, reliable and quantitative measurements. This dissertation is based on theoretical and experimental studies of silicon photonic sensing architectures in terms of sensor performance improvement and unit-cost reduction.Specifically, two novel sub-wavelength grating-based (SWG) waveguide configurations are presented to improve the sensitivity. Leveraging the advantage of SWG metamaterials, the substrate-overetch (SOE) and multi-box SWG devices present a largely extended modal size and surface contact area, which gives 10-time enhanced sensitivity compared to the conventional devices. In addition, by employing the Bragg grating as the sensing architecture, the multi-box SWG-based grating configuration achieves a lower detection limit compared to the microring resonator (MRR) counterpart and demonstrates the capability for monitoring small molecule interactions.Replacing the laser with a broadband source can provide a lower-cost solution for the optical system. Therefore, two cost-effective broadband light source-based sensing implementations are proposed and demonstrated with acceptable sensitivities. The first implementation uses cascaded MRRs for index monitoring, where the analyte variation is converted to the photocurrent change as the readout. The second implementation uses a phase-shifted Bragg grating-based symmetrical Mach-Zehnder interferometer, where the analyte variation maps the intensity change at the resonant wavelength. Furthermore, a system-level integration of active silicon photonic sensors using Fan-Out Wafer-Level-Packaging (FOWLP) is proposed in the dissertation, which can reduce the die size down to 1 mm² while simplifying the microfluidic and optical integration. Leveraging the CMOS foundries and the proposed FOWLP technique, the unit cost of each packaged sensing die can be reduced to several dollars.
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Enhancing the performance of silicon photonics biosensors (2017)
Silicon photonic biosensors have the potential to transform medical diagnostics and healthcare delivery. Hundreds of these nano-scale sensors can be integrated onto a single millimeter-sized silicon substrate. They are fabricated in established CMOS foundries leveraging similar economies-of-scale achieved by electronic integrated circuits. This also enables their potential integration with electronic read out circuitry on a single chip. As near-infrared light propagates through nanoscale silicon wires, a portion of the light resides outside the waveguide interacting with biomolecules on the waveguide’s surface. While silicon photonic biosensors have demonstrated performances approaching today’s gold-standard diagnostic, the enzyme-linked immunosorbent assay (ELISA), improving their performance expands the potential use for applications requiring higher sensitivities and detection limits. To this end, this thesis describes efforts to optimize established biosensor configurations and develop novel structures with performance that exceeds commercially available silicon photonic biosensor platforms. This involves improving the bulk and surface sensitivity, detection limit, and quality factor of transverse electric (TE) and magnetic (TM) mode resonators in various waveguide topologies. Specifically, TM mode microring resonators, microdisk resonators, thin waveguide resonators, and the first of its kind sub-wavelength grating microring resonator with a 10X sensitivity improvement over today’s commercially available ring resonators are presented. Furthermore the use novel TE mode slot-waveguide and TM mode strip waveguide Bragg gratings which facilitate higher sensitivities (8X) and lower detection limits for biosensing applications are described. Finally, suspended Bragg grating structures are investigated to further improve sensitivity. To support the design and characterization efforts required to efficiently investigate many different sensors, a testing platform and process design kit (PDK) was developed. The test platform automatically tests hundreds of devices and orchestrates complex, multi-hour assays. The PDK reduces first-time design risk and expedites chip testing. Both have been open-sourced and are in use by more than a dozen academic and commercial research groups in various countries.
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A Microfluidic Platform to Study Real-Time Tumour Cell and Tumour Spheroid Response to Chronic and Transient Hypoxia (2016)
Cell-based screening of cancer treatments is used early in the drug development process to test the efficacy and toxicity of treatment candidates prior to animal and human testing. Current cell-based screening methods offer limited predictive capacity, contributing to the high percentage of drugs that fail during the clinical trial stage (80-95% for cancer treatments). One shortcoming of traditional cell-based screening platforms is their inability to recreate many aspects of the natural environment of tumour cells, which can affect treatment response. One important aspect of the microenvironment that can affect cell behaviour and treatment response is oxygenation. Irregular blood vessel formation can cause tumour oxygenation to be much lower than that of surrounding tissue, and spatial and temporal variations in oxygen can be present. Temporal variations can occur at timescales up to several cycles/hour: changes that are too fast to recreate using standard technologies like well plates due to their long diffusion distances. This thesis presents a novel microfluidic platform to expose cells to both chronic and time-varying oxygen profiles and study their response. Microfluidics technology is combined with 3-D cell culture in tumour spheroids, which can better recreate other aspects of the tumour microenvironment (such as cell-cell and cell-matrix interactions) than traditional 2-D culture. The functionality of the oxygen control device is verified using both finite-element modelling and integrated optical oxygen sensors. Two novel methods for oxygen sensor microfabrication are presented, and the functionality of sensors during long-term experiments is studied. Precise oxygen control is demonstrated using the microfluidic system, with oxygen switching times of
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Modeling and Analysis of Diffusive Molecular Communication Systems (2015)
Diffusive molecular communication (MC) is a promising strategy for the transfer of information in synthetic networks at the nanoscale. If such devices could communicate, then it would expand their cumulative capacity and potentially enable applications such as cooperative diagnostics in medicine, bottom-up fabrication in manufacturing, and sensitive environmental monitoring. Diffusion-based MC relies on the random motion of information molecules due to collisions with other molecules. This dissertation presents a novel system model for three-dimensional diffusive MC where molecules can also be carried by steady uniform flow or participate in chemical reactions. The expected channel impulse response due to a point source of molecules is derived and its statistics are studied. The mutual information between consecutive observations at the receiver is also derived. A simulation framework that accommodates the details of the system model is introduced. A joint estimation problem is formulated for the underlying system model parameters. The Cramer-Rao lower bound on the variance of estimation error is derived. Maximum likelihood estimation is considered and shown to be better than the Cramer-Rao lower bound when it is biased. Peak-based estimators are proposed for the low-complexity estimation of any single channel parameter. Optimal and suboptimal receiver design is considered for detecting the transmission of ON/OFF keying impulses. Optimal joint detection provides a bound on detector performance. The weighted sum detector is proposed as a suboptimal alternative that is more physically realizable. The performance of a weighted sum detector can become comparable to that of the optimal detector when the environment has a mechanism to reduce intersymbol interference.A model for noise sources that continuously release molecules is studied. The time-varying and asymptotic impact of such sources is derived. The model for asymptotic noise is used to approximate the impact of multiuser interference and also the impact of older bits of intersymbol interference.
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Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Conceptualization and development of in vitro vascular models for studying Alzheimer's Disease (2022)
The human brain is an extraordinarily complex organ that is regulated by the input of nutrients and oxygen and the removal of waste via the vascular network. Understanding how the local microenvironment of cells that make up the neurovascular unit influences the health of the human brain could allow for better understanding of disease pathology, such as Alzheimer’s Disease (AD). Traditional animal-based models are limited in some of their physiological similarities with humans; therefore, human-based in vitro models are desirable. Standard cell culture is often performed in a 2-dimensional, static well plate, which lacks many of the physiological properties seen in the human brain. Moving towards in vitro models that include defined cellular architectures and includes flow on the endothelial cell layer could overcome some of the challenges associated with standard well plate models. In this thesis, in vitro models of the capillary and arteriole are conceptualized and developed. Two capillary-based microfluidic designs are developed, with a focus on the fabrication techniques used for the master molds, as well as the endothelial cell layer optimization. Having a tight endothelial cell layer is important to ensure that transport into and out of the brain is based on transcellular transport and not due to a leaky barrier. The first capillary model described includes a hydrogel-based extracellular matrix, and the second contains a planar membrane acting as a substrate for the endothelial cell barrier. In addition, this work highlights improvements to a previously used tissue chamber that contains a cell-laden scaffold. The motivation for these improvements includes the fragility of the tissue chamber and only being able to perform in-line sampling from the circulating fluid within the “blood” side, limiting the ability to perform in-line vessel transport studies. The fabrication of custom end-caps, to improve the tissue chamber stability, and the inclusion of sampling ports to the “brain” side are also described.
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Exploring the microfluidic organ-on-chip platform for aerosol exposure study (2022)
The microfluidic organ-on-chip platform exhibits promise for helping bridge the in vitro-in vivo gap through incorporating key elements of the in vivo microenvironment absent in traditional cell culture models. A prime example of such an element is fluid flow; tissues and their constituent cells are sensitive to a myriad of environmental stimuli, one of which is the mechanical forces that arise from wall shear stress exerted by these fluids. The work reported in the following thesis is focused on the small airways of the human lung and, particularly, how a microfluidic model of this physiology can be used in the study of aerosol exposure. Beginning with more traditional and well-established methods for the study of aerosol exposure in vitro, an aerosol generation apparatus was designed and employed to extract a panel of aerosols into solution for the assessment of cytotoxicity induced upon exposure in an airway epithelial cell line. A PDMS-based microfluidic small-airway-on-chip was, then, designed based on models reported in literature with optimizations included to balance physiological mimicry with established in vitro culture yields. A recirculating flow regime was developed, which enabled the application of wall-shear-stresses in ranges experienced by airway epithelia in vivo while minimizing consumption of reagents and maintaining the presence of excreted factors and chemokines. The microfluidic design and operating protocols were validated using both functional assays (permeability, inflammatory mediator release) and morphological features (F-actin localization). The small-airway-on-chip was, then, deployed in a novel experiment comprising the exposure of airway epithelium to aerosol generated from burnt wood at a dynamic ALI. Finally, an emerging method for characterization was explored: future applications of the silicon photonics platform to potential sensing and detection of biomolecules secreted by cells residing in organs-on-chip. Proof of concept experiments demonstrating the detection of recombinant interleukin 8 in buffer at natively expressed concentrations in vitro were performed.
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Developing a Novel Heterogeneous Three Electrode System for a PDMS-Based Microfluidic Electrochemical Sensor (2016)
Biosensors have seen an increased use in recent years as an in situ testing device for various industries such as agriculture, environmental sustainability, food, health, etc. On-site testing devices have an advantage over traditional testing system because they can be used for real-time monitoring and improving the accuracy of time-sensitive detection results. Out of all the industries, the health industry benefits most from in situ devices as point-of-care diagnostic devices. Point-of-care devices are useful tools to quickly diagnose diseases and direct patients’ course of treatment in low-income countries with few resources. In 2014 there were approximately 9.6 million global cases of tuberculosis (TB) and 1.5 million deaths due to TB (caused by the infection of Mycobacterium Tuberculosis [MTB]). Globally, this made TB the second most common cause of death by an infection disease in 2014. With a rising incidence of multi drug-resistant and extensive drug-resistant TB, TB is again becoming a global issue that wealthy countries will likely be unable to ignore. Since transmission is commonly through airborne particulates, early diagnosis and correct treatment of TB are fundamental to not only preventing the spread of the disease, but also eradicating it. Currently, the screening and detection tools that lower resource countries have are limited. Although the MTB genome has been sequenced since 1998, cost-effective, point-of-care, gene-based detection technology has had limited development. Since the integration of MEMS technology to biologically relevant needs in the late 90s there has been much development in creating small, portable detection systems for point-of-care use. Furthermore, this work details the development of a novel heterogeneous 3-material 3-electrode electrochemical sensor in a PDMS based bonded device. This sensor was developed with the intention of integration into a biosensor system. The final 3-electrode system was composed of 3 different materials: Au counter electrode, carbon working electrode, and Ag/AgCl reference electrode. The 3 material 3-electrode system was tested as an electrochemical system by detection of aqueous 5 μM carminic acid in room temperature and post 65◦C heating conditions. Parallel work was done to develop a robust, leak-free bonding method that survived 65◦C heating conditions and preserved electrochemical functionality.
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Development and evaluation of a novel approach to producing uniform 3-D tumor spheroid constructs (2016)
In vitro tumor spheroid models have been developed using microfluidic systems to generate 3-D hydrogel beads containing components of alginate and ECM protein, such as collagen, with high uniformity and throughput. During bead gelation, alginate acts as a fast gelling component helping to maintain the spherical shape of beads and to prevent adjacent or underlying beads from coalescing when working with the slower gelling temperature and pH-sensitivity of collagen components. There are also well-known limitations in using microfluidic systems when working with temperature-sensitive components of collagen type I, and it is determined that to produce uniform hydrogel droplets through a microfluidic system, the mixtures must be homogeneous. However, the issue of collagen’s sensitivity to temperature causes concern for chunks of collagen gel inside of the mixture before bead encapsulation; therefore causing the mixture to become non-uniform and risking chip clogging. In order to overcome this limitation, previous approaches have used a cooling system during bead encapsulation while tumor cells were also present in the mixture, but this procedure assisted in postponing collagen gelation prior to bead production and potentially contributing to a delay in cell proliferation.Here a novel yet simple method is developed to prepare homogeneous pre-bead-encapsulation-mixtures containing collagen through ultrasonication, while extending cell viability and proliferation. This method allows the cultivation of homogenous TS cultures with high uniformity and compact structure, and not only maintains cell viability but also stimulates the proliferation of cells in alginate/collagen hydrogel bead cultures. Depending on the sonication parameters, time and temperature, gelation of collagen is controlled by small sized fibrils to thick fibers. Human-source-Michigan-Cancer-Foundation-7 (MCF-7) cells isolated from a breast cancer cell line are successfully incorporated into alginate/collagen mixtures, followed by sonication, and then bead production. After bead gelation, the encapsulated MCF-7 cells remained viable and proliferated to form uniform TSs when the beads contained alginate and collagen. Results indicate that ultrasound treatment provides a powerful technique to change the structure of collagen from fiber to fibril, and to disperse collagen fibers in the mixture homogeneously for an application to generate uniform hydrogel beads and spheroids while not disturbing cell proliferation.
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Investigations into Inkjet Cell Printing Hydrodynamics through Microscopy Imaging Techniques (2015)
Inkjet bioprinting technology aims to accurately and precisely dispense biological materials in a spatially predefined pattern within a three-dimensional space. The technology has a multitude of applications in the biomedical field such as in drug discovery and tissue or organ engineering. However, there are known limitations in an inkjet nozzle's capabilities in dispensing cells as the cell ejection rate does not follow any predictable distributions. In this work, the cell behaviors within a piezoelectric nozzle due to droplet ejection were classified through high speed brightfield imaging. With each ejected droplet, one of three cell behaviors was observed to occur: cell travel, cell ejection, or cell reflection. Cell reflection is an undesirable phenomenon which may adversely affect an inkjet's capability to reliably dispense cells. To further study how the hydrodynamics within a nozzle can influence the cell's behavior, µPIV was performed to identify the flow field evolution during droplet ejection. Through the study of cell motion, it was observed that the viscosity of the media in the cell suspension plays an important role in influencing the cell behavior. This was experimentally studied with the tracking of cells within the inkjet nozzle in a higher viscosity 10% w/v Ficoll PM400 cell suspension. As hypothesized, the addition of Ficoll PM400 was effective in preventing the occurrence of cell reflection which promises to increase the reliability in inkjet bioprinting systems.
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A Three-Dimensional (3D) Defocusing-based Particle Tracking Method and Applications to Inertial Focusing in Microfluidic Devices (2014)
Three-dimensional analysis of particles in flows within microfluidic devices is a necessary technique in the majority of current microfluidics research. One method that allows for accurate determination of particle positions in channels is defocusing-based optical detection. This thesis investigates the use of the defocusing method for particles ranging in size from 2-18 μm without the use of a three-hole aperture. Using a calibration-based analysis motivated by previous work, we were able to relate the particle position in space to its apparent size in an image. This defocusing method was then employed in several studies in order to validate its effectiveness in a wide range of particle/flow profiles. An initial study of gravitational effects on particles in low Reynolds number flows was conducted, showing that the method is accurate for particles with sizes equal to or greater than approximately 2 μm. We also found that the resolution of particle position accuracy was within 1 μm of expected theoretical results. Further studies were conducted in inertial focusing conditions, where viscous drag and inertial lift forces balance to create unique particle focusing positions in straight channels. Steady-state inertial studies in both rectangular and cylindrical channel geometries showed focusing of particles to positions similar to previous work, further verifying the defocusing method. A new regime of inertial focusing, coined transient flow, was also investigated with the use of the 3D defocusing method. This study established new regimes of particle focusing due to the effects of a transient flow on inertial forces. Within the transient study, the effects of fluid and particle density on particle focusing positions were also investigated. Finally, we provide recommendations for future work on the defocusing method and transient flows, including potential applications.
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Alternative Replica Molding Methods for Polymer Based Microfluidic Channels (2014)
Microfluidics provides an opportunity to create low cost devices that can potentially contain many elements of a diagnostics lab on a single chip. While the cost of the finished product may be low, a common method of fabricating microfluidic devices such as soft lithography can be expensive to prototype due to the use of photolithography equipment meant for the semiconductor industry. In addition, the majority of microfluidic research has been done using rectangular channels but in some cases the ability to make circular cross-section channel microfluidic devices would be very useful. For areas such as modelling cardiovascular flows, investigating micro flow cytometry and inertial particle focusing, the ability to create circular channels could provide improvement over the use of rectangular channels. To address these issues, an ultra low cost method of making silicon molds patterned with SU-8 has been developed as well as a method to create circular microfluidic channels via hot embossing and double casting techniques in both thermoplastic materials and PDMS. This hot embossing based method to create round channels allows for the rapid creation of straight and curving round channels in PMMA and other plastics as well as a method to create PDMS round channels using soft lithography.
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Novel fabrication of flexible microelectrodes with macroporous platinum film using latex polystyrene sphere template (2014)
With great strides in neuroscience that have been made in the past decade, further understandings of complex neural systems require extensive neural information from chronic implantation of biocompatible neural devices. Polyimide-based flexible microelectrode arrays were one of the earlier biocompatible neural devices due to its mechanical impedance matching with brain tissue. In this work, we propose to incorporate non-conventional laser ablation method for fabrication of flexible biocompatible microelectrodes. We also present a novel approach to modifying flexible microelectrodes with macroporous platinum film using latex polystyrene sphere template. Maskless laser ablation was used to pattern the electrode, and probe definition as well creating the contact openings of flexible polyimide electrodes. Laser ablation is a non-photolithographic method which does not require conventional cleanroom environment and is ideal for rapid prototyping of devices. An ordered polystyrene bead template was deposited by simple pipetting of bead solution over gold contact openings and evaporating in ambient room setting. Pulsed-potentiostatic mode electrochemical deposition of platinum through the polystyrene bead template resulted in increase in effective surface area of electrodes. The impedance of the platinum modified electrodes increased by two orders of magnitude compared to unmodified electrodes. Synergetic modification of microelectrodes with macroporous platinum film and polymer-brush coating can lead to fabrication of highly biocompatible microelectrode with low impedance characteristics.
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Enhanced piezoelectric drop-on-demand cell dispensing through neutral buoyancy (2012)
Micro dispensing of cell based suspensions offers enhanced precision and accuracy for a wide variety of applications including high throughput cell based assays, single cell analyses and tissue engineering. One technology that offers such promises is piezoelectric inkjet printing. However, the small size scale of internal system features makes it difficult to eject particle based suspensions due to undesirable phenomena within the suspension. In particular, it has been hypothesized that inkjet dispensing of cell based suspensions has been hindered by sedimentation and aggregation of cells. The objective of this thesis was to investigate the phenomenon of cellular sedimentation and determine whether it affects piezoelectric dispensing reliability, and to devise a method to mitigate this phenomenon. The challenge in stabilizing suspensions to halt sedimentation lies in constraints involving cellular viability, and the rheological limitations imposed by the capability of the inkjet system itself. We achieved stabilized cellular suspensions through the use of a sucrose co-polymer, Ficoll-PM400. We show herein that stabilizing the suspension to be dispensed through this method greatly improves the reliability of the system over long periods of time, and increases the consistency of cell counts. Cell viability was shown to remain constant even when suspended for periods of 1 hour. The change in viscosity of the suspension also decreased the amount of clogging events that have hampered particle based inkjet dispensing approaches. Lastly, we give recommendations regarding future work on this technology.
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