Model and Appearance Based Analysis of Neuronal Morphology from Different Microscopy Imaging Modalities

The neuronal morphology analysis is key for understanding how a brain works. This process requires the neuron imaging system with single-cell resolution; however, there is no feasible system for the human brain. Fortunately, the knowledge can be inferred from the model organism, Drosophila melanogaster, to the human system. This dissertation explores the morphology analysis of Drosophila larvae at single-cell resolution in static images and image sequences, as well as multiple microscopy imaging modalities. Our contributions are on both computational methods for morphology quantification and analysis of the influence of the anatomical aspect. We develop novel model-and-appearance-based methods for morphology quantification and illustrate their significance in three neuroscience studies. Modeling of the structure and dynamics of neuronal circuits creates understanding about how connectivity patterns are formed within a motor circuit and determining whether the connectivity map of neurons can be deduced by estimations of neuronal morphology. To address this problem, we study both boundary-based and centerline-based approaches for neuron reconstruction in static volumes. Neuronal mechanisms are related to the morphology dynamics; so the patterns of neuronal morphology changes are analyzed along with other aspects. In this case, the relationship between neuronal activity and morphology dynamics is explored to analyze locomotion procedures. Our tracking method models the morphology dynamics in the calcium image sequence designed for detecting neuronal activity. It follows the local-to-global design to handle calcium imaging issues and neuronal movement characteristics. Lastly, modeling the link between structural and functional development depicts the correlation between neuron growth and protein interactions. This requires the morphology analysis of different imaging modalities. It can be solved using the part-wise volume segmentation with artificial templates, the standardized representation of neurons. Our method follows the global-to-local approach to solve both part-wise segmentation and registration across modalities. Our methods address common issues in automated morphology analysis from extracting morphological features to tracking neurons, as well as mapping neurons across imaging modalities. The quantitative analysis delivered by our techniques enables a number of new applications and visualizations for advancing the investigation of phenomena in the nervous system

Examining Uptake of Nanomaterials by Eukaryotic Cells with Digital Image Cytometry

Due to their small size and related interesting properties, artificial nanoma-terials are utilized for a great number of biological and medical applications. Cell entry routes, intracellular trafficking and processing of nanoparticles, which determine their fate, efficiency, and toxicity, are depending on various parameters of the specific nanomaterial, such as size, surface charge, surface chemistry and elasticity. Nanoparticle-cell interactions are typically elucidated by means of fluorescence microscopy. Cell functions can be observed by a multiplicity of commercially available probes. For the quantification of cell features from images (image cytometry), computer-based algorithms are favoured to avoid bias introduced by the subjective perception of the observer. By applying high throughput microscopy in combination with digital image cytometry the screening of high numbers of cells is made possible. With the large quantity of obtained data, cell populations can be identified and, in general, results that are statistically meaningful are obtained. In the first part of this work this method is applied in order to examine the cellular responses upon exposure to plasmonic poly(methacrylic acid)-coated gold nanoparticles (Au NPs) with respect to morphology and viability of human endothelial and epithelial cells (HUVECs and HeLa cells). Au NPs of 4-5 nm size were chosen which had been thoroughly characterized in terms of their physico-chemical parameters. These particles bear interesting properties for biomedical applications and, for several years, have been in the focus of research. In this work significant impacts on mitochondrial and lysosomal morphology upon exposure to the Au NPs are reported. The alteration of the structure of the cytoskeleton and a dramatically reduced proliferation are described. Interestingly, the smallest dose inducing the described cellular responses was of one or two magnitudes lower than those, where acute cytotoxicity and an increase in the production of reactive oxygen species (ROS) were observed. In the second part the process of endocytosis of polymer capsules is examined. These systems are seen as a promising tool for intracellular cargo delivery and release. After lipid raft-mediated phagocytosis, the capsules are transferred from the neutral extracellular medium to increasingly acidic intracellular vesicles. By embedding a pH-sensitive fluorescent dye into the cavity of the capsule the uptake process and the associated acidification can be monitored time-dependently. It is demonstrated that the kinetic of the acidification process strongly depends on the stiffness of the capsules. Soft particles with minor stiffness are transported faster into lysosomal structures than stiffer ones. Additionally, these sensor particles are used to confirm the importance of the V1G1-subunit of the vacuolar ATPase being responsible for vesicle acidification

Virtual infection modeling for Aspergillus fumigatus in human and murine alveoli

Der Der filamentöse pathogene Pilz Aspergillus fumigatus kann schwere Infektionen wie die invasive pulmonale Aspergillose in immungeschwächten Patienten verursachen. Verbunden mit einer hohen Mortalität und einer steigenden Inzidenz der letzten Jahrzehnte bezeugt dies die Notwendigkeit zur Erforschung seines opportunistischen Verhaltens sowie zur Entwicklung effizienter Behandlungsstrategien, um Menschenleben zu retten. Da die Lunge, als primäres Ziel von A. fumigatus Infektionen, nur begrenzt experimentell in vivo studiert werden kann, verfolgt diese Arbeit den Ansatz agenten-basierter Simulation. Die kumulative Dissertation basiert auf 4 veröffentlichten Manuskripten. Untersucht wurden dabei die Vergleichbarkeit von natürlichen Infektionen im Menschen und künstlichen Infektionen im etablierten Mausmodell. Eine zweite Veröffentlichung untersucht den Einfluss von Kohn'schen Poren auf die Dynamik der Immunabwehr gegen Aspergillus fumigatus. Eine dritte Veröffentlichung untersucht die Anwendbarkeit von dynamischen Kugeloberflächenfunktionen - Spherical Harmonics - als Werkzeug der Klassifikation und Beschreibung von beweglichen Zellen. Die vierte Veröffentlichung präsentiert erstmals einen Aspergillose Chip auf Mikrofluidikchips. Dies erlaubt es, die Pathogen-Wirt-Beziehungen unter realistischen Bedingungen zu untersuchen sowie das Wachstum der Pilzhyphen zu quantifizieren

Nanoparticles in medicine: Automating the analysis process of high-throughput microscopy data.

Automated tracking of cells across timelapse microscopy image sequences typically employs complex segmentation routines and/or bio-staining of the tracking objective. Often accurate identification of a cell's morphology is not of interest and the accurate segmentation of cells in pursuit of non-morphological parameters is complex and time consuming. This thesis explores the potential of internalized quantum dot nanoparticles as alternative, bio- and photo-stable optical markers for tracking the motions of cells through time. CdTe/ZnS core-shell quantum dots act as nodes in moving light display networks within A549, epithelial, lung cancer cells over a 40 hour time period. These quantum dot fluorescence sources are identified and interpreted using simplistic algorithms to find consistent, non-subjective centroids that represent cell centre locations. The presented tracking protocols yield an approximate 91% success rate over 24 hours and 78% over the full 40 hours. The nanoparticle moving light displays also provide simultaneous collection of cell motility data, resolution of mitotic traversal dynamics and identification of familial relationships enabling the construction of multi-parameter lineage trees. This principle is then developed further through inclusion of 3 different coloured quantum dots to create cell specific colour barcodes and reduce the number of time points necessary to successfully track cells through time. The tracking software and identification of parameters without detailed morphological knowledge is also demonstrated through automated extraction of DOX accumulation profiles and Cobalt agglomeration accruement statistics from two separate toxicology assays without the need for cell segmentation

Intracellular delivery and voltage sensitivity of nanomaterials for the optical imaging of neuronal activity

Monitoring the electrical signals generated by neurons to transmit information, is central to understanding how the brain and nervous systems work. The photoluminescence (PL) of some nanomaterials, such as semiconductor quantum dots (QDs) and fluorescent nanodiamonds (NDs), has shown higher sensitivity to electrical fields than that of any previously reported probes, which may address the persistent challenge of robust optical voltage imaging. The fundamental issue for implementing voltage-sensitive nanomaterials (VSM) in live neurons is their delivery into the plasma membrane bilayer. Currently, the delivery has been demonstrated on QDs via their spontaneous insertion directly into the plasma membrane bilayer, or indirectly into the bilayer of liposomes that later fuse with the plasma membrane. In both methods, QDs are introduced from the extracellular space and implemented to image the activity of neuronal assemblies. The first part of this thesis explores the implementation of VSM in another scenario, i.e., the voltage imaging from multiple sites on single neurons. After direct intracellular delivery, amphiphilic nanomaterials are expected to spread into distal processes and insert into the plasma membrane bilayer, being able to monitor the electrical activity in the smallest neuronal structures, such as dendritic spines. Here, the intracellular delivery of nontargeted QDs as an example, has been demonstrated by microelectrophoresis technique, where electrical currents were applied to eject charged QDs through fine-tipped glass micropipettes into living cells. The amount of delivered QDs was finely controlled by tuning the ejection duration, which had a substantial impact on preserving short-term and long-term cell health. Delivered QDs were homogeneously distributed throughout the cytoplasm and presented pure Brownian diffusion without endosomal entrapment. These original and promising results lay the foundation to apply the microelectrophoresis technique to other VSM, including the protocol for preparing nanomaterials suspension and the required tip sizes of micropipettes, which are key to their successful intracellular loading. Another fundamental issue is ascertaining the PL responses of these nanomaterials to applied voltage modulations. The second part of this thesis describes the fabrication of a multilayer device that can apply a homogeneous electric field to the embedded nanomaterials (NDs as an example). By using ultrasonication, NDs were well dispersed as single particles within the device, where the PL responses of individual NDs can be examined. Other fabrication details, such as film thickness and electrode deposition, were also described. These results provide a high-throughput screening platform to characterize the voltage sensitivities of different nanomaterials, which helps to iteratively improve their design and synthesis, including composition, size, shape, and band alignment. Collectively, the findings in this thesis provide a significant contribution to the unique interface of neuroscience and nanomaterials regarding the optical visualization of neuronal activity. The pioneering work here facilitates the future use of microelectrophoresis technique to deliver various VSM for multisite voltage imaging of single neurons. The deployment of the multilayer device promotes the development and optimization of new nanomaterials with enhanced voltage sensitivity. With these fundamental challenges to be addressed in the near future, real-time in vivo voltage imaging may be attainable in relevant animal models to elucidate the complex function of brain and nervous systems.Thesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 202

Investigating the biological impacts of nanoengineered materials in Caenorhabditis elegans and in vitro

In nematode Caenorhabditis elegans, the chronic and multi-generational toxicological effects of commercially relevant engineered nanoparticles (ENPs), such as quantum dots (QDs) and silver (AgNP) caused significant changes in a number of physiological endpoints. The increased water-solubility of ENPs in commercial products, for example, makes them increasingly bioavailable to terrestrial organisms exposed to pollution and waste in the soil. Since 2008, attention to the toxicology of nanomaterials in C. elegans continues to grow. Quantitative data on multiple physiological endpoints paired with metal analysis show the uptake of QDs and AgNPs, and their effects on nematode fitness. First, C. elegans were exposed for four generations through feeding to amphiphilic polymer coated CdSe/ZnS (core-shell QDs), CdSe (core QDs), and different sizes of AgNPs. These ENPs were readily ingested. QDs were qualitatively imaged in the digestive tract using a fluorescence microscopy and their and AgNP uptake quantitatively measured using ICP-MS. Each generation was analyzed for changes in lifespan, reproduction, growth and motility using an automated computer vision system. Core-shell QDs had little impact on C. elegans due to its metal shell coating. In contrast, core QDs lacked a metal shell coating, which caused significant changes to nematode physiology. In the same way, at high concentrations of 100 ppm, AgNP caused the most adverse effect to lifespan and reproduction related to particle size, but its adverse effect to motility had no correlation to particle size. Using C. elegans as an animal model allowed for a better understanding of the negative impacts of ENPs than with cytotoxicity tests. Lastly, to test the toxicity of water-dispersed fullerene (nanoC60) using human dermal fibroblast cells, this thesis investigated a suite of assays and methods in order to establish a standard set of cytotoxicity tests. Ten assays and methods assessed nanoC60 samples of different purities to show differences in cytotoxic effects. Washed samples of fullerenes, with negligible traces of THF and other impurities, rendered the solution nontoxic. Even when exposed to UV-irradiation, washed nanoC60 were not photosensitized and did not cause cellular death. This work characterizes ENPs and investigates their impact in C. elegans and cells to assess toxicity risks to the environment and to human health

Imaging studies of peripheral nerve regeneration induced by porous collagen biomaterials

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2013.Cataloged from PDF version of thesis.Includes bibliographical references.There is urgent need to develop treatments for inducing regeneration in injured organs. Porous collagen-based scaffolds have been utilized clinically to induce regeneration in skin and peripheral nerves, however still there is no complete explanation about the underlying mechanism. This thesis utilizes advanced microscopy to study the expression of contractile cell phenotypes during wound healing, a phenotype believed to affect significantly the final outcome. The first part develops an efficient pipeline for processing challenging spectral fluorescence microscopy images. Images are segmented into regions of objects by refining the outcome of a pixel-wide model selection classifier by an efficient Markov Random Field model. The methods of this part are utilized by the following parts. The second part extends the image informatics methodology in studying signal transduction networks in cells interacting with 3D matrices. The methodology is applied in a pilot study of TGFP signal transduction by the SMAD pathway in fibroblasts seeded in porous collagen scaffolds. Preliminary analysis suggests that the differential effect of TGFP1 and TGFP3 to cells could be attributed to the "non-canonical" SMADI and SMAD5. The third part is an ex vivo imaging study of peripheral nerve regeneration, which focuses on the formation of a capsule of contractile cells around transected rat sciatic nerves grafted with collagen scaffolds, 1 or 2 weeks post-injury. It follows a recent study that highlights an inverse relationship between the quality of the newly formed nerve tissue and the size of the contractile cell capsule 9 weeks post-injury. Results suggest that "active" biomaterials result in significantly thinner capsule already 1 week post-injury. The fourth part describes a novel method for quantifying the surface chemistry of 3D matrices. The method is an in situ binding assay that utilizes fluorescently labeled recombinant proteins that emulate the receptor of , and is applied to quantify the density of ligands for integrins a113, a2p1 on the surface of porous collagen scaffolds. Results provide estimates for the density of ligands on "active" and "inactive" scaffolds and demonstrate that chemical crosslinking can affect the surface chemistry of biomaterials, therefore can affect the way cells sense and respond to the material.by Dimitrios S. Tzeranis.Ph. D

Modulation of transglutaminase 2 by pituitary adenylyl cyclase activating polypeptide and nerve growth factor in neuroblastoma cells: a role in cell survival and neurite outgrowth

Several studies have reported that the multifunctional enzyme tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. Recently, TG2 enzymatic activity has been shown to be regulated by protein kinases such as PKA and PKC, modulating through G-protein coupled receptors (GPCRs). However, the regulation of TG2 following stimulation of GPCRs coupled to PKC and PKA activation are not fully understood. In neuronal cells, the neurotrophic factors; pituitary adenylate cyclase activating polypeptide (PACAP) and nerve growth factor (NGF) promote the differentiation, maturation, neurite outgrowth and survival of neurons via their GPCR's; the pituitary adenylate cyclase type 1 receptor (PAC1) and the tyrosine kinase receptor (TrKA) including multiple protein kinase signalling pathways. As some of these kinase pathways are associated with modulation of intracellular TG2 activity, it is conceivable to hypothesised that PACAP and NGF directly regulates TG2 activity. If this was found to be the case, it sought to determine whether the PACAP and/or NGF-induced neurite outgrowth and modulation of neuronal cell survival involved TG2. The main aims of this study were to investigate the molecular mechanisms underlying TG2 modulation by PACAP and NGF and its role in neurite outgrowth and neuroprotection in differentiating mouse N2a and human SHSY5Y neuroblastoma cells. In the first part, the regulation of TG2 transamidase activity by PACAP-27 and NGF in differentiating mouse N2a and human SH-SY5Y neuroblastoma cells was assessed. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of iotin-X-cadaverine using confocal microscopy. TG2 phosphorylation was monitored via immunoprecipitation and Western blotting. In the second part of this study, the role of TG2 in PACAP-27 and NGF-induced cytoprotection was investigated by monitoring hypoxia-induced cell death and assessing MTT reduction, LDH release, and caspase-3 activity. The neurite outgrowth was assessed using high-throughput analysis of immunofluorescently stained cells. In differentiating N2a and SH-SY5Y cells, stimulation of PAC1 and TrKA receptor with PACAP-27 and NGF respectively, resulted in a time- and concentration- dependent activation of TG2 amine incorporation and protein crosslinking activity, which was abolished by TG2 inhibitors Z-DON and R283. Responses to PACAP-27 were attenuated mainly by pharmacological inhibition of protein kinase A (KT 5720 and Rp-cAMPs), MEK1/2 (PD 98059), PKB (Akt inhibitor XI) and by removal of extracellular Ca2+. Responses to NGF were abolished by MEK1/2 (PD 98059), PKB (Akt inhibitor XI) and PKC (Ro 31-8220). Immunoprecipitation technique showed that both PACAP-27 and NGF triggered the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by inhibition of MEK1/2 and PKA for PACAP and MEK1/2, PKB, PKC and removal of extracellular Ca2+ for NGF. Fluorescence microscopy demonstrated that PACAP-27 and NGF induced in situ TG2 activity. TG2 inhibition blocked PACAP-27- and NGF-induced attenuation of hypoxia-induced cell death and neurite outgrowth. Taken together, this study highlights for the first time the importance of TG2 in the cellular functions of PACAP and NGF in neuronal cells and provides initial characterisation of the molecular mechanisms possibly involved