Elevated eosinophil counts in the circulation and/or tissues is considered a clinical feature and biomarker of several chronic airway diseases including asthma. As such, many therapeutic biologics for asthma developed within the past decade target eosinophil recruitment to and accumulation in the airways to mixed success. Although the nature of adhesive interactions and directional migration of eosinophils has been well studied, there remains a lack of comprehensive understanding regarding the components which modulate eosinophil movement from the blood into respiratory tissues that impacts the efficacy of these clinical studies; therefore, continued research in this area may reveal novel therapeutic targets and ultimately improve clinical outcomes of patients with eosinophilia-mediated diseases. The Janssen lab serendipitously discovered that the mere perfusion of standard media without pharmacological additives over human eosinophils in vitro induced the release of intracellular calcium (Ca2+) reminiscent of chemokine-induced Ca2+ release well documented in the literature. The central focus of my doctoral research was to characterize this novel phenomenon of the perfusion-induced calcium response (PICR), and to determine its physiological role in the eosinophil extravasation process to inflamed tissue sites. In our first research objective, we optimized a protocol of eosinophil isolation directly from whole blood with emphases on maximizing population purity and yield efficiency while minimizing cell activation that could potentially interfere with secondary functional assays. For our latter two studies, we utilized real-time fluorescent confocal microscopy and immunofluorescence staining to investigate the PICR. We observed that the latency to the PICR post-perfusion was significantly shorter in eosinophils subjected to physiological rates of shear stress, suggesting a temporal-regulatory function of eosinophil mechanosensitivity. Furthermore, the disruption of the PICR via pharmacological inhibitors significantly reduced eosinophil motility by increasing the latency to cytoskeletal rearrangements (flattening onto substrate-coated surfaces, formation of membrane protrusions that explore the environment) necessary for cell migration out of the vasculature. Detailing the role of eosinophil sensitivity to the mechanical trigger of fluid shear stress expands upon the current paradigm of eosinophil recruitment and will contribute to the development of clinical strategies. / Dissertation / Candidate in Philosophy

<p>The interface between two dissimilar materials often presents a challenge for materials engineers. Mismatches of moduli, coefficients of thermal expansion, surface energies and chemical functionalities can create headaches for engineers seeking to control and understand interfacial bonding. In this work, I am interested in two specific interfacial problems: the adhesion of pressure sensitive adhesive tapes to various substrates and the interface in polymer reinforced composite materials between the reinforcement phase and the matrix.</p> <p>Pressure sensitive adhesive tapes (PSATs) are an important class of materials with applications ranging from medical adhesives to roadway markings. In this work, I present a novel 90° peel fixture to be used in the evaluation of road tapes on roadway surfaces in construction zones. This modular fixture was validated on control surfaces before demonstrating the capability to test pavement marking tapes from road surfaces. Within the context of medical adhesives, I am interested in the deformation of the skin around the PSAT during peeling. By developing a model to predict this deformation, adhesives can be tailored to mitigate skin damage. I present experimental evidence indicating the independence of peeling force to the elastic modulus of the substrate along with deformation measurements of skin analogs during the removal of a medical tape. A new model for predicting the deformation of soft substrates during peel is reported based on the contact mechanics of a rectangular prism indenting an elastic half space.</p> <p>Polymer matrix composites are another category of materials which are increasingly adopted to improve performance or efficiency by reducing the weight of components. These materials offer a high specific strength but often fail catastrophically rather than gradually. Using stress responsive fluorescent molecules called mechanophores, I present a methodology to quantify stresses within the polymeric matrix near the reinforcement phase. By correlating in situ fluorescence intensity measurements during a uniaxial tensile test to stresses predicted from a finite element analysis model, a calibration was developed. This calibration was then applied to increasingly complex composite geometries. Chemically bonding these mechanophores to the interface between two materials allows for the detection of interfacial failures through fluorescence microscopy. I present a technique to synthesize interfacial spirolactam mechanophores on industrially relevant epoxy and silica material systems. I demonstrate the ability of these systems to detect failures in the system through in situ confocal microscopy during deformation.</p>

The retinal pigment epithelium (RPE) is a transporting epithelium with polarized membrane domains. A unique characteristic of these cells is that their apical surface does not face a lumenal space, but is directly apposed to a layer of neurons (photoreceptors) and their associated extracellular matrix. Because the interaction occurring at this site is important for retinal attachment and particle phagocytosis, an attempt was made to identify epithelial molecules which potentially could mediate cell-cell or cell-matrix adhesion. In the present report, the subcellular localization of β1-integrins, the main receptors for extracellular matrix ligands, has been examined within Xenopus RPE. Several previously characterized antibodies were used in this analysis including: two rabbit polyclonal antibodies directed against purified chick muscle fibronectin receptor (pAbs No. 3818 and No. 2999), and a monoclonal antibody specific for Xenopus β1-integrin subunit (mAb 8C8). In Western blots of whole epithelial cell extracts, each of the antibodies intensely labeled a 115 kDa band, consistent with β1-integrin reactivity. One of the reagents (pAb No. 3818) also weakly stained unidentified bands of 50 and 100 kDa. Pre-clearing experiments demonstrated that pAb No. 3818 and mAb 8C8 both recognize the same detergent-soluble integrin: when cell extracts were depleted of β1-integrin by immunoprecipitation with mAb 8C8, the 115 kDa antigen recognized by pAb No. 3818 was not observed. Consistent with their similar immunochemical reactivities, each of the antibodies produced equivalent immunocytochemical staining of many eyecup tissues, including extraocular skeletal muscle cells, scleral and choroidal fibroblasts and vascular endothelium of the choroid and neural retina. In the native RPE, and isolated sheets of epithelium, however, qualitative differences in labeling between these antibodies were evident. Analysis by confocal microscopy showed that, while all three antibodies stained the basal surface of the epithelium, pAb No. 3818 also strongly labeled the apical microvillar surface. As the adjacent photoreceptors did not cross-react with this antibody in control experiments, the apical RPE staining could not be accounted for as contamination with retinal tissues during isolation. Furthermore, when the apical cell surface was selectively biotinylated in situ, and biotinylated proteins precipitated by streptavidin-agarose, β1-integrin was detected by immunoblotting with both mAb 8C8 and pAb No. 3818. This domain-specific material, however, represented only a fraction of the whole cell surface integrin: substantially greater amounts of tagged molecules could be detected when isolated epithelial sheets were biotinylated, most likely representing the basal protein. Based on these results, it can be concluded that β1-integrin is present in both basal and apical RPE plasma membranes. Molecules present in the apical, membrane may represent components of adhesion receptors responsible for retina-epithelium interactions.

Yes / Corneal images can be acquired using confocal microscopes which provide detailed images of the different layers inside the cornea. Most corneal problems and diseases occur in one or more of the main corneal layers: the epithelium, stroma and endothelium. Consequently, for automatically extracting clinical information associated with corneal diseases, or evaluating the normal cornea, it is important also to be able to automatically recognise these layers easily. Artificial intelligence (AI) approaches can provide improved accuracy over the conventional processing techniques and save a useful amount of time over the manual analysis time required by clinical experts. Artificial neural networks (ANN) and adaptive neuro fuzzy inference systems (ANFIS), are powerful AI techniques, which have the capability to accurately classify the main layers of the cornea. The use of an ANFIS approach to analyse corneal layers is described for the first time in this paper, and statistical features have been also employed in the identification of the corneal abnormality. An ANN approach is then added to form a combined committee machine with improved performance which achieves an accuracy of 100% for some classes in the processed data sets. Three normal data sets of whole corneas, comprising a total of 356 images, and seven abnormal corneal images associated with diseases have been investigated in the proposed system. The resulting system is able to pre-process (quality enhancement, noise removal), classify (whole data sets, not just samples of the images as mentioned in the previous studies), and identify abnormalities in the analysed data sets. The system output is visually mapped and the main corneal layers are displayed. 3D volume visualisation for the processed corneal images as well as for each individual corneal cell is also achieved through this system. Corneal clinicians have verified and approved the clinical usefulness of the developed system especially in terms of underpinning the expertise of ophthalmologists and its applicability in patient care.

Microporous carbonates host a significant portion of the remaining oil-in-place in the giant carbonate reservoirs of the Middle East. Improved understanding of petrophysical and multi-phase flow properties at the pore-scale is essential for the development of better oil recovery processes. These properties strongly depend on the 3D geometry and connectivity of the pore space. In this study, we harnessed the unique capabilities of fluorescence confocal laser scanning microscopy (CLSM) to capture both macroporosity and microporosity, down to 0.1 µm, to provide a more representative 3D representation of pore space compared to traditional methods. The experimental procedure developed was specifically designed to enable highresolution confocal 3D imaging of the pore space of carbonate systems. The protocol aims to render carbonates more "transparent" to CLSM by imaging etched epoxy pore casts of the sample and minimizing CLSM signal scattering. The resulting highquality 3D images of the multi-scale pore space allow more reliable petrophysical interpretation and prediction of transport properties. Additionally, we present a robust pore imaging approach that correlates 2D images produced by scanning electron microscopy (SEM) with the 3D models produced by CLSM that cover a range of scales, from millimeters in 3D to micrometers in 2D. For the first time, multi-color fluorescence confocal imaging was employed to characterize the geometric attributes of a porous medium. We foresee that the protocol developed in this study could be used as a standard protocol for obtaining high-quality 3D images of epoxy pore casts using confocal microscopy, and could contribute to improved characterization of micritic carbonate reservoirs and oil recovery methods. We also demonstrate the advantages of multi-scale and multi-color confocal images in realizing more accurate evaluations of petrophysical properties. Finally, we demonstrate that micro 3D printing (two-photon polymerization) can potentially be used to fabricate micromodels with sufficient resolution to capture the geometric attributes of micritic carbonates and that can replicate the inherent 3D interconnectivity between macro- and micro-pores.

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<p>This report primarily focuses on effectively obtaining a Stimulated Emission Depletion fluorescence (STED) microscope, while using Fluorescence Correlation Spectroscopy (FCS) as a guide for the alignment of the system. STED is a super-resolution microscopy technique that has gained favour in the biological sciences due to its ability to successfully resolve sub-diffraction structures within live cells. Moreover the ease with which it can be combined with FCS has extended the applications of this technique to the study of the dynamics within a system as well. The central premise of this work focuses around building a STED-FCS system and developing an alignment tool for obtaining a symmetric STED doughnut. Since the point spread functions (PSF) seen in confocal microscopy can be generally approximated by a Gaussian function, we approximate the doughnut PSF with a difference of Gaussian functions. We calculated an autocorrelation function (ACF) corresponding to the simplified Gaussian form of the doughnut PSF and we found that this ACF contained three very similar diffusion times, all inversely proportional to the dye diffusion coefficient. In agreement with the fact that the doughnut PSF is spread out compared to the purely Gaussian PSF, the doughnut ACF amplitude is lower and its average diffusion time large. Lastly we calculated the quality factor, which is the product of the amplitude of the correlation function with the average intensity, Q=G(0)*I, for the purposes of alignment of the system. When translating the confocal pinhole along an axis of the doughnut we were able to identify the centre of the doughnut due to the presence of a minimum in Q which can be very handy for alignment of the doughnut with respect to the pinhole. This operation is essential when aligning the excitation and STED beam. For future work, a road map for alignment of the two beams in the focal plane is also presented utilizing the cross correlation function between the two beams.</p> / Master of Science (MSc)

The number of food borne illnesses associated with the consumption of fresh fruits and vegetables and their minimally processed products (juices) has increased over the past years. Of particular interest is the ability of microbial pathogens to internalize and survive in fresh produce that are commonly used for juices. This research project addresses the issue of the ability of Escherichia coli O157:H7 to internalize and survive in whole apples before and after harvest. Four cultivars of apples, Redfree, Red Delicious, Golden Delicious, and York, were inoculated under field conditions with a surrogate strain of E. coli, Escherichia coli ATCC 25922. The Redfree cultivar was inoculated at the beginning of its growth stage (day 0), and again 30 days later, and sampled for two weeks, until E. coli was not recoverable through microbiological methods after three successive sampling days. Red Delicious, Golden Delicious, and York cultivars were spray inoculated with the surrogate strain two weeks before their anticipated harvest date and sampled every other day until E. coli was not recoverable for three successive sampling days. For each cultivar, the presence of E. coli ATCC 25922 was not detectable after 7 to 9 days. In the laboratory study the Red Delicious, Golden Delicious, Rome, and York cultivars received one of three treatments; unblemished control, bruising, or puncturing. The apples were inoculated by immersion in cold water containing E. coli O157:H7 GFP, incubated for three days then microbiologically analyzed for presence of the bacteria. In all cases, the punctured apples of each cultivar showed the greatest uptake of E. coli O157:H7 GFP. Escherichia coli O157:H7 GFP was visualized in flesh and core sections of untreated, bruised, and punctured apples of all cultivars. The microbe was found in between cells, but not within cells of the apple. Internalization of Escherichia coli in whole apples on the tree is not likely, and leads to the conclusion that internalization is a post-harvest problem. Internalization may occur before pressing or processing of apples, leading to an increased risk of infection with E. coli for consumers of apple products that are not properly treated to destroy pathogens. Internalization does occur when apples are immersed in solutions containing the pathogen Escherichia coli O157:H7, and better post harvest controls need to be implemented in order to prevent this in whole apples that are used for cider and juice production. / Master of Science

Après une introduction brève de l’historique de l’imagerie dermatologique non invasive, ce travail est divisé 3 parties. 1) Présentation d’un projet de développement d’un tomographe à cohérence optique miniaturisé, peu onéreu devant permettre une diffusion de cette technique aux dermatologues exerçant en dehors des hôpitaux. Il s’agi d’un projet ANR DOCT-VCSEL Portable Optical Coherence Tomography with MEMS-VCSEL swept- sources for skin analysis ANR 2015 / Défi sociétal « Vie, Santé et Bien-Etre » Axe 13 « Technologies pour la santé » 2) Présentation d’un projet dont le but est l’identification de lésions cutanées cancéreuses au moyen d’un nouvel OCT haute définition développé par la société DAMAE, issue de l’Institut supérieur d’Optique de Palaiseau. Il s’agit d’un dispositif qui sera dans un premier temps réservé aux centre d’excellence en imagerie dermatologique. 3) la reprise des 52 publications ayant trait à l’imagerie cutanée auxquelles j’ai participé et référencées dans les bases de données internationales au 31 décembre 2016. Ce travail couvre l’ensemble de l’imagerie non invasive dermatologique moderne et aborde des sujets qui n’avaient jamais été étudié de la sorte. Notamment les muqueuses et l’appareil oculaire antérieur mais aussi l’identification par microscopie confocale des marge chirurgicales ou l’association microscopie confocale spectrométrie Raman / After a brief introduction to the history of non-invasive dermatological imaging, this work is divided into 3 parts. 1) Presentation of a project for the development of a low-cost miniaturized optical coherence tomograph to allow dissemination of this technique to dermatologists practicing outside hospitals. This is an ANR project: DOCT-VCSEL Portable Optical Coherence Tomography with MEMS-VCSEL swept-sources for skin analysis ANR 2015 / Societal Challenge "Life, Health and Welfare" Axis 13 “Technologies for Health" 2) Presentation of a project whose goal is the identification of cancer skin lesions by means of a new high definition OCT developed by the company DAMAE, resulting from the Higher Institute of Optics of Palaiseau. It is a device that will initially be reserved for centers of excellence in dermatological imaging. 3) Presentation of 52 publications related to skin imaging, in which I participated, and referenced in the international databases as of December 31, 2016. This work covers all modern dermatological non-invasive imaging and addresses Subjects that had never been studied in this way. Notably the mucous membranes and the anterior ocular apparatus but also the identification by confocal microscopy of the surgical margins or the association confocal microscopy Raman spectrometry