Doctor of Philosophy

dissertationElectron microscopy can visualize synapses at nanometer resolution, and can thereby capture the fine structure of these contacts. However, this imaging method lacks three key elements: temporal information, protein visualization, and large volume reconstruction. For my dissertation, I developed three methods in electron microscopy that overcame these limitations. First, I developed a method to freeze neurons at any desired time point after a stimulus to study synaptic vesicle cycle. Second, I developed a method to couple super-resolution fluorescence microscopy and electron microscopy to pinpoint the location of proteins in electron micrographs at nanometer resolution. Third, I collaborated with computer scientists to develop methods for semi-automated reconstruction of nervous system. I applied these techniques to answer two fundamental questions in synaptic biology. Which vesicles fuse in response to a stimulus? How are synaptic vesicles recovered at synapses after fusion? Only vesicles that are in direct contact with plasma membrane fuse upon stimulation. The active zone in C. elegans is broad, but primed vesicles are concentrated around the dense projection. Following exocytosis of synaptic vesicles, synaptic vesicle membrane was recovered rapidly at two distinct locations at a synapse: the dense projection and adherens junctions. These studies suggest that there may be a novel form of ultrafast endocytosis

Reconstruction of the neuromuscular junction connectome

Motivation: Unraveling the structure and behavior of the brain and central nervous system (CNS) has always been a major goal of neuroscience. Understanding the wiring diagrams of the neuromuscular junction connectomes (full connectivity of nervous system neuronal components) is a starting point for this, as it helps in the study of the organizational and developmental properties of the mammalian CNS. The phenomenon of synapse elimination during developmental stages of the neuronal circuitry is such an example. Due to the organizational specificity of the axons in the connectomes, it becomes important to label and extract individual axons for morphological analysis. Features such as axonal trajectories, their branching patterns, geometric information, the spatial relations of groups of axons, etc. are of great interests for neurobiologists in the study of wiring diagrams. However, due to the complexity of spatial structure of the axons, automatically tracking and reconstructing them from microscopy images in 3D is an unresolved problem. In this article, AxonTracker-3D, an interactive 3D axon tracking and labeling tool is built to obtain quantitative information by reconstruction of the axonal structures in the entire innervation field. The ease of use along with accuracy of results makes AxonTracker-3D an attractive tool to obtain valuable quantitative information from axon datasets

Detection of multiple innervation zones from multi-channel surface EMG recordings with low signal-to-noise ratio using graph-cut segmentation

Knowledge of the location of muscle Innervation Zones (IZs) is important in many applications, e.g. for minimizing the quantity of injected botulinum toxin for the treatment of spasticity or for deciding on the type of episiotomy during child delivery. Surface EMG (sEMG) can be noninvasively recorded to assess physiological and morphological characteristics of contracting muscles. However, it is not often possible to record signals of high quality. Moreover, muscles could have multiple IZs, which should all be identified. We designed a fully-automatic algorithm based on the enhanced image Graph-Cut segmentation and morphological image processing methods to identify up to five IZs in 60-ms intervals of very-low to moderate quality sEMG signal detected with multi-channel electrodes (20 bipolar channels with Inter Electrode Distance (IED) of 5 mm). An anisotropic multilayered cylinder model was used to simulate 750 sEMG signals with signal-to-noise ratio ranging from -5 to 15 dB (using Gaussian noise) and in each 60-ms signal frame, 1 to 5 IZs were included. The micro- and macro- averaged performance indices were then reported for the proposed IZ detection algorithm. In the micro-averaging procedure, the number of True Positives, False Positives and False Negatives in each frame were summed up to generate cumulative measures. In the macro-averaging, on the other hand, precision and recall were calculated for each frame and their averages are used to determine F1-score. Overall, the micro (macro)-averaged sensitivity, precision and F1-score of the algorithm for IZ channel identification were 82.7% (87.5%), 92.9% (94.0%) and 87.5% (90.6%), respectively. For the correctly identified IZ locations, the average bias error was of 0.02±0.10 IED ratio. Also, the average absolute conduction velocity estimation error was 0.41±0.40 m/s for such frames. The sensitivity analysis including increasing IED and reducing interpolation coefficient for time samples was performed. Meanwhile, the effect of adding power-line interference and using other image interpolation methods on the deterioration of the performance of the proposed algorithm was investigated. The average running time of the proposed algorithm on each 60-ms sEMG frame was 25.5±8.9 (s) on an Intel dual-core 1.83 GHz CPU with 2 GB of RAM. The proposed algorithm correctly and precisely identified multiple IZs in each signal epoch in a wide range of signal quality and is thus a promising new offline tool for electrophysiological studies.The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the Seventh Framework Programme of the European Union (FP7/2007-2013) under REA grant agreement no. 600388 (TECNIOspring programme), from the Agency for Business Competitiveness of the Government of Catalonia, ACCIÓ, and from Spanish Ministry of Economy and Competitiveness- Spain (project DPI2014-59049-R).Peer ReviewedPostprint (published version

Artificial Intelligence, Mathematical Modeling and Magnetic Resonance Imaging for Precision Medicine in Neurology and Neuroradiology

La tesi affronta la possibilità di utilizzare metodi matematici, tecniche di simulazione, teorie fisiche riadattate e algoritmi di intelligenza artificiale per soddisfare le esigenze cliniche in neuroradiologia e neurologia al fine di descrivere e prevedere i patterns e l’evoluzione temporale di una malattia, nonché di supportare il processo decisionale clinico. La tesi è suddivisa in tre parti. La prima parte riguarda lo sviluppo di un workflow radiomico combinato con algoritmi di Machine Learning al fine di prevedere parametri che favoriscono la descrizione quantitativa dei cambiamenti anatomici e del coinvolgimento muscolare nei disordini neuromuscolari, con particolare attenzione alla distrofia facioscapolo-omerale. Il workflow proposto si basa su sequenze di risonanza magnetica convenzionali disponibili nella maggior parte dei centri neuromuscolari e, dunque, può essere utilizzato come strumento non invasivo per monitorare anche i più piccoli cambiamenti nei disturbi neuromuscolari oltre che per la valutazione della progressione della malattia nel tempo. La seconda parte riguarda l’utilizzo di un modello cinetico per descrivere la crescita tumorale basato sugli strumenti della meccanica statistica per sistemi multi-agente e che tiene in considerazione gli effetti delle incertezze cliniche legate alla variabilità della progressione tumorale nei diversi pazienti. L'azione dei protocolli terapeutici è modellata come controllo che agisce a livello microscopico modificando la natura della distribuzione risultante. Viene mostrato come lo scenario controllato permetta di smorzare le incertezze associate alla variabilità della dinamica tumorale. Inoltre, sono stati introdotti metodi di simulazione numerica basati sulla formulazione stochastic Galerkin del modello cinetico sviluppato. La terza parte si riferisce ad un progetto ancora in corso che tenta di descrivere una porzione di cervello attraverso la teoria quantistica dei campi e di simularne il comportamento attraverso l'implementazione di una rete neurale con una funzione di attivazione costruita ad hoc e che simula la funzione di risposta del modello biologico neuronale. E’ stato ottenuto che, nelle condizioni studiate, l'attività della porzione di cervello può essere descritta fino a O(6), i.e, considerando l’interazione fino a sei campi, come un processo gaussiano. Il framework quantistico definito può essere esteso anche al caso di un processo non gaussiano, ovvero al caso di una teoria di campo quantistico interagente utilizzando l’approccio della teoria wilsoniana di campo efficace.The thesis addresses the possibility of using mathematical methods, simulation techniques, repurposed physical theories and artificial intelligence algorithms to fulfill clinical needs in neuroradiology and neurology. The aim is to describe and to predict disease patterns and its evolution over time as well as to support clinical decision-making processes. The thesis is divided into three parts. Part 1 is related to the development of a Radiomic workflow combined with Machine Learning algorithms in order to predict parameters that quantify muscular anatomical involvement in neuromuscular diseases, with special focus on Facioscapulohumeral dystrophy. The proposed workflow relies on conventional Magnetic Resonance Imaging sequences available in most neuromuscular centers and it can be used as a non-invasive tool to monitor even fine change in neuromuscular disorders and to evaluate longitudinal diseases’ progression over time. Part 2 is about the description of a kinetic model for tumor growth by means of classical tools of statistical mechanics for many-agent systems also taking into account the effects of clinical uncertainties related to patients’ variability in tumor progression. The action of therapeutic protocols is modeled as feedback control at the microscopic level. The controlled scenario allows the dumping of uncertainties associated with the variability in tumors’ dynamics. Suitable numerical methods, based on Stochastic Galerkin formulation of the derived kinetic model, are introduced. Part 3 refers to a still-on going project that attempts to describe a brain portion through a quantum field theory and to simulate its behavior through the implementation of a neural network with an ad-hoc activation function mimicking the biological neuron model response function. Under considered conditions, the brain portion activity can be expressed up to O(6), i.e., up to six fields interaction, as a Gaussian Process. The defined quantum field framework may also be extended to the case of a Non-Gaussian Process behavior, or rather to an interacting quantum field theory in a Wilsonian Effective Field theory approach

Doctor of Philosophy

dissertationNeuroscientists are developing new imaging techniques and generating large volumes of data in an effort to understand the complex structure of the nervous system. The complexity and size of this data makes human interpretation a labor intensive task. To aid in the analysis, new segmentation techniques for identifying neurons in these feature rich datasets are required. However, the extremely anisotropic resolution of the data makes segmentation and tracking across slices difficult. Furthermore, the thickness of the slices can make the membranes of the neurons hard to identify. Similarly, structures can change significantly from one section to the next due to slice thickness which makes tracking difficult. This thesis presents a complete method for segmenting many neurons at once in two-dimensional (2D) electron microscopy images and reconstructing and visualizing them in three-dimensions (3D). First, we present an advanced method for identifying neuron membranes in 2D, necessary for whole neuron segmentation, using a machine learning approach. The method described uses a series of artificial neural networks (ANNs) in a framework combined with a feature vector that is composed of image and context; intensities sampled over a stencil neighborhood. Several ANNs are applied in series allowing each ANN to use the classification context; provided by the previous network to improve detection accuracy. To improve the membrane detection, we use information from a nonlinear alignment of sequential learned membrane images in a final ANN that improves membrane detection in each section. The final output, the detected membranes, are used to obtain 2D segmentations of all the neurons in an image. We also present a method that constructs 3D neuron representations by formulating the problem of finding paths through sets of sections as an optimal path computation, which applies a cost function to the identification of a cell from one section to the next and solves this optimization problem using Dijkstras algorithm. This basic formulation accounts for variability or inconsistencies between sections and prioritizes cells based on the evidence of their connectivity. Finally, we present a tool that combines these techniques with a visual user interface that enables users to quickly segment whole neurons in large volumes

Automated Reconstruction of Dendritic and Axonal Trees by Global Optimization with Geometric Priors

We present a novel probabilistic approach to fully automated delineation of tree structures in noisy 2D images and 3D image stacks. Unlike earlier methods that rely mostly on local evidence, ours builds a set of candidate trees over many different subsets of points likely to belong to the optimal tree and then chooses the best one according to a global objective function that combines image evidence with geometric priors. Since the best tree does not necessarily span all the points, the algorithm is able to eliminate false detections while retaining the correct tree topology. Manually annotated brightfield micrographs, retinal scans and the DIADEM challenge datasets are used to evaluate the performance of our method. We used the DIADEM metric to quantitatively evaluate the topological accuracy of the reconstructions and showed that the use of the geometric regularization yields a substantial improvement

Microvasculopathy in SMA is driven by a reversible autonomous endothelial cell defect

Spinal muscular atrophy (SMA) is a neuromuscular disorder due to degeneration of spinal cord motor neurons caused by deficiency of the ubiquitously expressed SMN protein. Here, we present a retinal vascular defect in patients, recapitulated in SMA transgenic mice, driven by failure of angiogenesis and maturation of blood vessels. Importantly, the retinal vascular phenotype was rescued by early, systemic SMN restoration therapy in SMA mice. We also demonstrate in patients an unfavorable imbalance between endothelial injury and repair, as indicated by increased circulating endothelial cell counts and decreased endothelial progenitor cell counts in blood circulation. The cellular markers of endothelial injury were associated with disease severity and improved following SMN restoration treatment in cultured endothelial cells from patients. Finally, we demonstrated autonomous defects in angiogenesis and blood vessel formation, secondary to SMN deficiency in cultured human and mouse endothelial cells, as the underlying cellular mechanism of microvascular pathology. Our cellular and vascular biomarker findings indicate microvasculopathy as a fundamental feature of SMA. Our findings provide mechanistic insights into previously described SMA microvascular complications, and highlight the functional role of SMN in the periphery, including the vascular system, where deficiency of SMN can be addressed by systemic SMN-restoring treatment

AutoGraff: towards a computational understanding of graffiti writing and related art forms.

The aim of this thesis is to develop a system that generates letters and pictures with a style that is immediately recognizable as graffiti art or calligraphy. The proposed system can be used similarly to, and in tight integration with, conventional computer-aided geometric design tools and can be used to generate synthetic graffiti content for urban environments in games and in movies, and to guide robotic or fabrication systems that can materialise the output of the system with physical drawing media. The thesis is divided into two main parts. The first part describes a set of stroke primitives, building blocks that can be combined to generate different designs that resemble graffiti or calligraphy. These primitives mimic the process typically used to design graffiti letters and exploit well known principles of motor control to model the way in which an artist moves when incrementally tracing stylised letter forms. The second part demonstrates how these stroke primitives can be automatically recovered from input geometry defined in vector form, such as the digitised traces of writing made by a user, or the glyph outlines in a font. This procedure converts the input geometry into a seed that can be transformed into a variety of calligraphic and graffiti stylisations, which depend on parametric variations of the strokes

Zebrafish as a Model to Understand the Impact of Inactivity and Neuromuscular Electrical Stimulation on Duchenne Muscular Dystrophy

Skeletal muscle plasticity is imperative for functional adaptation to changing demands in activity. Although a great deal is known about the structural and functional plasticity of healthy skeletal muscle, far less is known about plasticity in diseased muscle. Here, we combined the power of the zebrafish model with the adaptability of neuromuscular electrical stimulation (NMES) to study the basic mechanisms of plasticity in the zebrafish model of Duchenne Muscular Dystrophy (DMD). Four NMES paradigms, defined by their frequency, delay, and voltage, were designed to emulate the repetition and load schemes of human resistance training programs. Additionally, two inactivity paradigms were designed to emulate activity patterns in individuals with DMD. Three sessions of endurance NMES improve muscle structure, increase swim velocity and distance traveled, and extend survival. Endurance NMES significantly increased the number and length of branching for neuromuscular junctions. Nuclear surface area and volume also significantly increased following endurance NMES. Time-lapse imaging suggests less degeneration and improved regeneration of the fast-twitch muscle fibers. Conversely, three days of inactivity worsen muscle structure and decreases survival. Strikingly, inactivity followed by a single session of endurance or power NMES obliterates muscle resilience. Therefore, our data clearly indicate that, at least in the zebrafish model, some resistance training is beneficial whereas inactivity is deleterious for dystrophic muscle. More importantly, though, our data provide a new methodology with which to study muscle plasticity in healthy and diseased muscle

Investigating human Schwann cell phenotypes and outcome measures of muscle reinnervation

Peripheral Nerve Injury (PNI) often causes partial or complete paralysis and/or loss of sensation of the segment of the body involved. Traumatic PNI is a global problem and can result in significant disability and socio-economic impacts. Clinical translation of new therapeutics for the treatment of PNI is challenged by the little information that is known about the cellular and molecular features that underpin human nerve regeneration. Moreover, clinical models and measurements that can quantify the efficacy of new treatments for PNI are not well established. Therefore, this PhD explored injured and healthy human nerve samples liberated from reconstructive nerve procedures to characterise the cellular and molecular features of human peripheral nerve degeneration. Associated with this theme of characterisation of human nerve injury, the recovery of motor units in reinnervated elbow flexor muscles following nerve transfer was quantified using Motor Unit Number Estimation (MUNE). In order to better understand the relationship of MUNE with the biological process of nerve regeneration, an animal model of nerve injury was used to investigate the association between MUNE and histological markers of regeneration. MUNE was found to be a sensitive marker of muscle reinnervation in human and animal models of nerve regeneration. Moreover, MUNE demonstrated a correlation with histological markers of muscle reinnervation. It is known that these changes in the number of motor units are accompanied by changes in muscle volume. Therefore, using the same surgical scenario of nerve transfer to reanimate elbow flexor muscles, this PhD measured the recovery of muscle volume following nerve transfer to reanimate elbow flexor muscles using quantitative Magnetic Resonance Imaging (MRI) techniques. It was found that MRI assessment of muscle volume is a measure that is sensitive to the biological process of nerve regeneration. With further data, this has the capacity to determine the efficacy of new therapeutics for the treatment of PNI and predict the likely functional recovery following PNI. In summary, the findings represent an important step towards understanding the in vivo cellular and molecular events in human nerve degeneration. In addition, MUNE and quantitative MRI techniques were found to represent sensitive and responsive measures of nerve regeneration. With further data, the findings presented here will help new therapeutic options for human nerve injury advance