Microfluidic device with 3D electrode structure for high throughput dielectrophoretic applications

Cataloged from PDF version of thesis.Includes bibliographical references (leaves 55-62).Thesis (M.S.): Bilkent University, The Department of Mechanical Engineering, İhsan Doğramacı Bilkent University, 2014.Microfluidics is the combination of micro/nano fabrication techniques together with knowledge of fluid behavior at the microscopic level to pursue powerful techniques in controlling, manipulating and measuring chemical, physical and biological processes at micro/nano scale. Sorting and separation of bio-particles are highly considered in diagnostics and biological analyses. By implementing the characteristics of microscale flow phenomenon, dielectrophoresis (DEP) has offered unique advantages for microfluidic devices. In DEP devices asymmetric pair of planar or three dimensional (3D) electrodes could be employed to generate non-uniform electric field. In DEP applications, facing 3D sidewall electrodes is considered to be the key solution of increasing device throughput because of producing homogeneous electric fields along the height of microchannels. Despite all advantages, fabrication of 3D vertical electrodes requires considerable challenge. In this thesis, in order to highlight the advantage of 3D electrodes over planar electrodes, the simulations are performed. Based on the developed computational model, the design parameters are decided. For the fabrication of the device, two different fabrication techniques have been proposed. In the first method, both the mold and the electrodes are fabricated using high precision machining. In the second method, the mold is fabricated with tilted sidewalls using high precision machining and the electrodes are deposited on the sidewall using sputtering together with a shadow mask fabricated using wire electric discharge machining (WEDM). The both techniques are assessed as highly repeatable and robust methods. Only the manipulation of particles with negative-DEP has been demonstrated in the experiments, and the throughput values up to 105 particles/min have been reached in a continuous flow.by Soheila Zeinali.M.S

Modeling and simulation of EGF-CSF-1 pathway to investigate Glioma - Macrophage interaction in brain tumors

Glioblastoma multiform (GBM) is one of the most lethal forms of brain cancers. The biggest difficulties for diagnostics and treatment of GBM underlie in its dynamic and complex macro and microenvironment. Glioma cells, stromal cells and tumor-associated immune cells (microglia/macrophage-TAMs) become a complex tissue with their physical and chemical communication network. TAMs are the predominant infiltrating immune cells in malignant GBMs and stimulate tumor invasion, angiogenesis, and metastasis. The epidermal growth factor (EGF) and colony stimulating factor 1 (CSF-1) paracrine signaling loop plays a key role in communication between glioma cells and TAMs. We developed a mathematical model to investigate macrophage-glioma cell interactions using CSF-1 and EGF paracrine-acting agents. Our model presents change of EGF and CSF-1 concentration both on the surfaces of cells and within a well-defined tumor microenvironment, in a domain, with respect to interaction time and distance between TAMs and glioma cells. Our simulation results confirm that from low-grade glioma to high-grade glioma, concentration of CSF-1 increases both on the surfaces of macrophages and within the domain. Therefore, reproduction and adsorption of CSF-1 correlates with the grade of malignancy in human gliomas, which is a good agreement with recent findings

Remodeling of an in vitro microvessel exposed to cyclic mechanical stretch.

In the lung, vascular endothelial cells experience cyclic mechanical strain resulting from rhythmic breathing motions and intraluminal blood pressure. Mechanical stress creates evident physiological, morphological, biochemical, and gene expression changes in vascular endothelial cells. However, the exact mechanisms of the mechanical signal transduction into biological response remain to be clarified. Besides, the level of mechanical stress is difficult to determine due to the complexity of the local distension patterns in the lung and thus assumed to be the same as the one acting on the alveolar epithelium. Existing in vitro models used to investigate the effect of mechanical stretch on endothelial cells are usually limited to two-dimensional (2D) cell culture platforms, which poorly mimic the typical three-dimensional structure of the vessels. Therefore, the development of an advanced in vitro vasculature model that closely mimics the dynamic of the human lung vasculatures is highly needed. Here, we present the first study that investigates the interplay of the three-dimensional (3D) mechanical cyclic stretch and its magnitude with vascular endothelial growth factor (VEGF) stimulation on a 3D perfusable vasculature in vitro. We studied the effects of the cyclic strain on a perfusable 3D vasculature, either made of human lung microvascular endothelial cells or human umbilical vein endothelial cells embedded in a gel layer. The in vitro 3D vessels underwent both in-vivo-like longitudinal and circumferential deformations, simultaneously. Our results showed that the responses of the human lung microvascular endothelial cells and human umbilical vein endothelial cells to cyclic stretch were in good agreement. Although our 3D model was in agreement with 2D model in predicting a cytoskeletal remodeling in response to different magnitudes of cyclic stretch, however we observed several phenomena in 3D model that 2D model was unable to predict to. Angiogenic sprouting induced by VEGF decreased significantly in presence of cyclic stretch. Similarly, while treatment with VEGF increased vascular permeability, the cyclic stretch restored vascular barrier tightness and significantly decreased vascular permeability. One of the major findings of this study was that a 3D microvasculature can be exposed to a much higher mechanical cyclic stress level than reported in the literature without any dysfunction of its barrier. For higher magnitudes of the cyclic stretch, the applied longitudinal strain level was 14% and the associated circumferential strain reached the equivalent of 63%. In sharp contrast with our findings, such strain typically leads to the disruption of the endothelial barrier in a 2D stretching assay and is considered pathological. This highlights the importance of 3D modeling to investigate mechanobiology effects rather than using a simple endothelial monolayer which truly recapitulates the in vivo situation

Dystrophic Epidermolysis Bullosa: COL7A1 Mutation Landscape in a Multi-Ethnic Cohort of 152 Extended Families with High Degree of Customary Consanguineous Marriages

Dystrophic epidermolysis bullosa is a heritable skin disease manifesting with sub-lamina densa blistering, erosions, and chronic ulcers. COL7A1, encoding type VII collagen, has been identified as the candidate gene for dystrophic epidermolysis bullosa. In this study, we have identified COL7A1 mutations in a large multi-ethnic cohort of 152 extended Iranian families with high degree of consanguinity. The patients were diagnosed by clinical manifestations, histopathology, and immunoepitope mapping. Mutation detection consisted of a combination of single nucleotide polymorphism-based whole-genome homozygosity mapping, Sanger sequencing, and gene-targeted next-generation sequencing. A total of 104 distinct mutations in COL7A1 were identified in 149 of 152 families (98%), 56 (53%) of them being previously unreported. Ninety percent of these mutations were homozygous recessive, reflecting consanguinity in these families. Three recurrent mutations were identified in five or more families, and haplotype analysis suggested a founder effect in two of them. In conclusion, COL7A1 harbored mutations in the overwhelming majority of patients with dystrophic epi-dermolysis bullosa, and most of them in this Iranian cohort were consistent with autosomal recessive inheri-tance. The mutation profile attests to the impact of consanguinity in these families

Gene-Targeted Next Generation Sequencing Identifies PNPLA1 Mutations in Patients with a Phenotypic Spectrum of Autosomal Recessive Congenital Ichthyosis: The Impact of Consanguinity

Heritable forms of ichthyoses, also referred to as generalized Mendelian disorders of cornification, are phenotypically a highly heterogeneous group of conditions caused by mutations in a number of genes playing a role in keratinocyte differentiation and epidermal barrier function (Baden and Digiovanna, 2013; Schmuth et al., 2013). These diseases are characterized by scaling and hyperkeratosis with associated cutaneous and extracutaneous features. This group of disorders is also genetically heterogeneous, with autosomal dominant, autosomal recessive, and X-linked inheritance being described. A specific subgroup of inherited ichthyoses is the autosomal recessive congenital ichthyosis (ARCI), with many newborns presenting as collodion babies, but the subsequent clinical presentation and the spectrum of severity can be highly variable (Richard and Bale, 2014). In the most severe forms, such as harlequin ichthyosis, the disease is often fatal during the early postnatal period, whereas at the other end of the continuum of the spectrum, the disease may present with a relatively mild scaling and variable degree of palmoplantar keratoderma. There is considerable genetic heterogeneity in ARCI, and as many as nine different genes are known to harbor biallelic mutations; these include TGM1, ALOXE3, ALOX12B, NIPAL4, ABCA12, CYP4F22, PNPLA1, LIPN, and CERS3. Previous reports have suggested that mutations in TGM1 account for 30e65% of patients with ARCI, whereas mutations in LIPN, PNPLA1, and CERS3 have been reported only in a few consanguineous families (Richard and Bale, 2014). With the advent of next generation sequencing (NGS), there has been tremendous progress in facilitating the mutation detection in various heritable skin disorders, including ichthyosis (South et al., 2015; Takeichi et al., 2013). In fact, at least 38 different genes have now been suggested to be associated with the ichthyotic phenotypes, either as the primary mutated genes or modifying the phenotypic presentation. To elucidate the genetic basis of ichthyosis in Iran, a country of approximately 80 million people with high prevalence of customary consanguineous marriages, we developed a gene-targeted NGS array consisting of 38 genes reported in association with ichthyosis phenotypes. Identification of specific mutations in a large number of families has allowed us to examine phenotype/genotype correlations with respect to both intra- and interfamilial heterogeneity, in part because of extensive consanguinity in these families. In this study, we identified six distinct and, to our knowledge, previously unreported mutations in the PNPLA1 gene in nine families

Immune cell extravasation in an organ-on-chip to model lung inflammation.

Acute respiratory distress syndrome (ARDS) is a severe lung condition with high mortality and various causes, including lung infection. No specific treatment is currently available and more research aimed at better understanding the pathophysiology of ARDS is needed. Most lung-on-chip models that aim at mimicking the air-blood barrier are designed with a horizontal barrier through which immune cells can migrate vertically, making it challenging to visualize and investigate their migration. In addition, these models often lack a barrier of natural protein-derived extracellular matrix (ECM) suitable for live cell imaging to investigate ECM-dependent migration of immune cells as seen in ARDS. This study reports a novel inflammation-on-chip model with live cell imaging of immune cell extravasation and migration during lung inflammation. The three-channel perfusable inflammation-on-chip system mimics the lung endothelial barrier, the ECM environment and the (inflamed) lung epithelial barrier. A chemotactic gradient was established across the ECM hydrogel, leading to the migration of immune cells through the endothelial barrier. We found that immune cell extravasation depends on the presence of an endothelial barrier, on the ECM density and stiffness, and on the flow profile. In particular, bidirectional flow, broadly used in association with rocking platforms, was found to importantly delay extravasation of immune cells in contrast to unidirectional flow. Extravasation was increased in the presence of lung epithelial tissue. This model is currently used to study inflammation-induced immune cell migration but can be used to study infection-induced immune cell migration under different conditions, such as ECM composition, density and stiffness, type of infectious agents used, and the presence of organ-specific cell types

Hypotrichosis with juvenile macular dystrophy: Combination of whole-genome sequencing and genome-wide homozygosity mapping identifies a large deletion in CDH3 initially undetected by whole-exome sequencing-A lesson from next-generation sequencing.

BACKGROUND: Hypotrichosis with juvenile macular dystrophy (HJMD) is an autosomal recessive disorder characterized by abnormal growth of scalp hair and juvenile macular degeneration leading to blindness. We have explored the genetic basis of HJMD in a large consanguineous family with 12 affected patients, 1-76 years of age, with characteristic phenotypes. METHODS: We first applied genome-wide homozygosity mapping to 10 affected individuals for linkage analysis to identify the genomic region of the defective gene. All affected individuals shared a 7.2 Mb region of homozygosity on chromosome 16q21-22.3, which harbored 298 genes, including CDH3, previously associated with HJMD. However, whole-exome sequencing (WES) failed to identify the causative mutation in CDH3. RESULTS: Further investigation revealed a missense variant in a gene closely linked to CDH3 (1.4 Mb distance: FHOD1: c.1306A\u3eG, p.Arg436Gly). This variant was homozygous in all affected individuals and heterozygous in 18 out of 19 obligate carriers. While this variant was found by bioinformatics predictions to be likely pathogenic, a knock-in mouse for this variant, made by the CRISPR/Cas, showed no disease phenotype. However, using whole-genome sequencing (WGS), we were able to identify a novel Alu recombination-mediated deletion in CDH3:c.del161-811_246 + 1,044. CONCLUSION: WGS was able to identify a deep intronic deletion mutation, not detected by WES

The sustainability challenge of product information quality in the design and construction of facades : lessons from the Grenfell Tower fire

Purpose: This paper explores the quality and flow of facade product information and the capabilities for avoiding the risk of facade fires early in the design process. Design/methodology/approach: A qualitative case study using the process tracing method is conducted in two stages. First, a thematic analysis of reports and literature identified two categories for the problems that caused fast fire spread across the Grenfell Tower facade. This enabled classifying the identified problems into four stages of a facade life cycle: product design and manufacturing, procurement, facade design and construction. Second, the capabilities for avoiding the problems were explored by conducting in-depth interviews with 18 experts in nine countries, analyzing design processes and designers' expertise and examining the usability of three digital interfaces in providing required information for designing fire-safe facades. Findings: The results show fundamental flaws in the quality of facade product information and usability of digital interfaces concerning fire safety. These flaws, fragmented design processes and overreliance on other specialists increase the risk of design defects that cause fast fire spread across facades. Practical implications: The findings have implications for standardization of building product information, digitalization in industrialized construction and facade design management. Originality/value: This research adds to the body of knowledge on sustainability in the built environment. It is the first study to highlight the fundamental problem of facade product information, which requires urgent attention in the rapid transition toward digital and industrialized construction

Simulation of an integrated microfluidic device for bioparticle wash separation and concentration

Washing, separation and concentration of bioparticles are key operations for many biological and chemical analyses. In this study, the simulation of an integrated microfluidic device is studied. The proposed device has the capability to wash the bioparticles (transferring the bioparticles from one buffer solution to another), to separate the particles based on their dielectric properties and to concentrate the bioparticles. Washing and concentration of bioparticles are performed by acoustophoresis and the separation is performed by dielectrophoresis. For simulating the flow within the microchannel, a computational fluid dynamics model using COMSOL Multiphysics software is implemented. In order to simulate the particle trajectories under ultrasonic and electric field, point-particle assumption is chosen using MATLAB software. To account for the size variation of the bioparticles, particles with normal size distributions are used in-side the microchannel. The effect of the key design parameters such as flow rate, applied voltage etc. on the performance of the device is discussed