Modifications of perineuronal nets to regulate plasticity Heleen Merel van 't Spijker Perineuronal nets (PNNs) are macromolecular structures formed by neurons after closure of critical periods of plasticity. During development, the central nervous system (CNS) goes through critical periods of plasticity; a period when substantial changes occur to adapt to the environment, during which many synapses are formed and also discarded. When a region of the CNS has finished its development and reached an efficient neuronal circuit, the capacity for plasticity needs to be reduced to preserve the formed circuit. PNNs are formed around neurons during this period of reduced plasticity. PNNs consist of a backbone of hyaluronan, bound by chondroitin sulfate proteoglycans (CSPGs). Here, I present my studies on the possible modifications of PNNs to regulate plasticity. Firstly, I have investigated the potential use of 4-methylumbelliferone (4-MU) to reduce PNN formation in vivo. 4-MU reduces the formation of hyaluronan. Since hyaluronan is the backbone of PNNs, I hypothesized 4-MU treatment would reduce PNN formation. For this study, I developed a method to orally administer 4-MU to rats. Subsequently, I investigated whether 4-MU treatment can improve recovery of rats after spinal cord injury, both with behavioural tests and with immunohistochemistry. Secondly, I have investigated a new binding partner of PNNs, neuronal pentraxin 2 (Nptx2). Nptx2 is secreted by neurons and regulates AMPA receptor diffusion. Nptx2 knockout mice show a prolonged critical period of plasticity in the visual cortex. Here, I have identified Nptx2 as a new binding partner of PNNs. Nptx2 is found in isolated PNN protein preparations and is removed from the surface of neurons by digestion of PNNs with chondroitinase ABC. I also determined Nptx2 facilitates PNN formation in vitro. Addition of Nptx2 to the medium of cortical neurons leads to an increase of neurons that start to form PNNs, as well as an increase in size and density of PNNs. These findings indicate Nptx2 may be used as a modulator of PNNs. Thirdly, I investigated the interaction between Nptx2 and PNNs. I developed a sandwich ELISA to determine which glycan chains from PNNs bind to Nptx2. Nptx2 binds to chondroitin sulfate E and hyaluronan. To investigate the binding properties of Nptx2, I performed quartz crystal microbalance with dissipation monitoring for Nptx2 films. Furthermore, I developed crystals of purified Nptx2 and hyaluronan for x-ray crystallography. The here presented results provide new insights in potential approaches to modulate PNN formation. Both lines of research provide a further understanding of the factors which regulate PNNs and may allow for the development of treatments for PNN related disorders.

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abstract: Human-inhabited or -disturbed areas pose many unique challenges for wildlife, including increased human exposure, novel challenges, such as finding food or nesting sites in novel structures, anthropogenic noises, and novel predators. Animals inhabiting these environments must adapt to such changes by learning to exploit new resources and avoid danger. To my knowledge no study has comprehensively assessed behavioral reactions of urban and rural populations to numerous novel environmental stimuli. I tested behavioral responses of urban, suburban, and rural house finches (Haemorhous mexicanus) to novel stimuli (e.g. objects, noises, food), to presentation of a native predator model (Accipiter striatus) and a human, and to two problem-solving challenges (escaping confinement and food-finding). Although I found few population-level differences in behavioral responses to novel objects, environment, and food, I found compelling differences in how finches from different sites responded to novel noise. When played a novel sound (whale call or ship horn), urban and suburban house finches approached their food source more quickly and spent more time on it than rural birds, and urban and suburban birds were more active during the whale-noise presentation. In addition, while there were no differences in response to the native predator, rural birds showed higher levels of stress behaviors when presented with a human. When I replicated this study in juveniles, I found that exposure to humans during development more accurately predicted behavioral differences than capture site. Finally, I found that urban birds were better at solving an escape problem, whereas rural birds were better at solving a food-finding challenge. These results indicate that not all anthropogenic changes affect animal populations equally and that determining the aversive natural-history conditions and challenges of taxa may help urban ecologists better understand the direction and degree to which animals respond to human-induced rapid environmental alterations. / Dissertation/Thesis / Doctoral Dissertation Biology 2018

La motivation principale de cette thèse est d’être capable de prédire précisément la rupture d’une tôle d’aluminium anisotrope avec un chemin de déformation linéaire et non-linéaire. Dans le cas présent, le matériau utilisé est l’AA6016 et le chemin de déformation considéré est traction uniaxiale suivie de pliage jusqu’à rupture. Deux approches sont appliquées et comparées, l’une utilise la plasticité associée (AFR) et l’autre la plasticité non-associée (NAFR). Dans le but d’obtenir une bonne représentation de l’anisotropie en AFR, un critère de plasticité très flexible est utilisé : Yld2004-18p. L’identification des paramètres est faite avec une approche inverse qui consiste à minimiser itérativement l’écart entre les résultats numériques et expérimentaux. Une fois que l’écart arrête d’évoluer (minimum local) ou atteint une valeur prédéterminé e assez faible, l’optimisation s’arrête et les derniers paramètres mis à jour sont enregistrés. En corrélation avec des travaux de recherche plus récents, un modèle NAFR est utilisé pour modéliser l’anisotropie du matériau. Il combine deux critères de plasticité qui sont utilisés pour la surface d’écrouissage et le potentiel plastique. Leurs paramètres sont identifiés grâce aux ratios de contraintes et aux valeurs r obtenues expérimentalement. Concernant la rupture, des modèles découplés macroscopiques sont étudiés : un critère Hosford-Coulomb modifié et un critère basé sur DF2014. Ces deux critères prennent en compte les trois invariants du tenseur des contraintes pour prédire la déformation équivalente à rupture mais sont identifiés avec différentes méthodes pour prendre en compte l’anisotropie de la rupture. Enfin, des résultats sur des instabilités plastiques obtenus avec un modèle NAFR sont présentés dans le but de prouver les possibilités de cette approche comparée à une approche AFR. / The main motivation of this thesis is to be able to predict accurately the fracture of an anisotropic aluminium alloy thin sheet under linear and non-linear strain paths. In the studied case, the material used is the AA6016 and the non-linear strain path considered is uniaxial tension followed by free bending until fracture. Two approaches are considered and compared which respectively use the associated flow rule (AFR) and the non-associated flow rule (NAFR). In order to obtain a good representation of the high anisotropy of the material in AFR, a very flexible yield criterion is used: Yld2004-18p. The identification of its parameters is done with an inverse approach consisting of iteratively minimizing the gap between numerical and experimental results. Once this gap stops evolving (local minimum) or reaches a low enough pre-determined value, the optimization stops and the last updated parameters are saved. In correlation with more recent research work, a NAFR model is used to model the anisotropy of the material. It combines two different yield functions that are used for the yield surface and the plastic potential. Their parameters are identified by using stress ratios and rvalues measured experimentally. Concerning the fracture, uncoupled macroscopic models are studied: a modified Hosford-Coulomb and a DF2014 based criteria. Both these criteria take into account the three invariants of the stress tensor to predict the equivalent strain to fracture but their parameters are identified with different methods to take into account the anisotropy of the fracture. Finally, results on plastic instabilities obtained with a NAFR model are presented in order to prove the possibilities of this approach compared to AFR.

To experience the world, we depend on the ability of our brains to process information. Problems can occur when communication between neurons is not regulated, and a significant enough loss of stability could lead to conditions such as migraine and epilepsy. Homeostatic plasticity is thought to constrain activity within physiologically useful ranges. Our lab uses the fruit fly neuromuscular junction as a model synapse to study homeostatic plasticity. Homeostatic potentiation and homeostatic depression are two forms of homeostatic synaptic plasticity. Expression of a dominant negative glutamate receptor subunit in the muscle impairs its sensitivity to glutamate and triggers an increase in the number of vesicles released per evoked potential, or quantal content. This increase in quantal content is called homeostatic potentiation. We found that homeostatic potentiation is a reversible process: quantal content returns to normal levels when expression of the dominant negative ceases. We additionally found that homeostatic potentiation can be ablated at high temperature. Overexpression of the Vesicular Glutamate transporter (VGlut) causes an increase in the amplitude of spontaneous events, leading to a corresponding decrease in quantal content, called homeostatic depression. It is unknown to what degree homeostatic potentiation and homeostatic depression may share regulatory machinery. We screened genes required for homeostatic potentiation in the neuron for additional roles in homeostatic depression. We found that certain genes involved in calcium regulation, such as the IP3 receptor and ryanodine receptor, showed a substantial decrease in evoked potential amplitude in a VGlut overexpression background.

Les lymphocytes T régulateurs Foxp3+ jouent un rôle crucial dans l'établissement de la tolérance au soi, le contrôle des réponses inflammatoires et le maintien de l'homéostasie du système immunitaire. La compréhension des mécanismes moléculaires impliqués dans la fonction de ces cellules représente un défi important. Chez le rat, la déficience en Themis1, une nouvelle molécule de la signalisation du TCR, associée à un locus de 117kb d'origine BN induit un défaut fonctionnel des Tregs et le développement spontané d'une maladie inflammatoire des intestins. Au sein de ce locus, le rat BN présente deux polymorphismes non-synonymes, un au niveau du gène C3 et un au niveau du gène Vav1 (R63W). Ce dernier est un candidat potentiel du fait du rôle joué par Vav1 dans l'activation des lymphocytes T et de sa régulation par Themis1. Dans ce travail de thèse, j'ai étudié l'effet de la déficience en Themis1 associé au polymorphisme R63W de Vav1 chez la souris de fond génétique C57BL/6 sur les fonctions des lymphocytes T régulateurs. J'ai montré que la déficience en Themis1 associé au polymorphisme R63W induit un défaut fonctionnel des Tregs in vitro et in vivo dans un modèle de colite. Ce défaut est associé à une production accrue de cytokines pro-inflammatoires par ces Tregs. J'ai également mis en évidence que l'association de ces deux mutations induit une sensibilité accrue à la colite induite par le DSS. Au niveau moléculaire, j'ai mis en evidence que ce défaut fonctionnel est associé à une réduction de la signalisation du TCR impliquant les vois Erk et NF-ĸB. De plus, j'ai montré que l'inhibition d'une phosphatase de la signalisation du TCR, SHP-1, permet de restaurer les fonctions suppressives des Tregs Vav1R63W-Themis1-/-. Cette étude souligne l'importance de l'intégrité du hub de la signalisation impliquant Vav1, Themis1 et SHP-1 dans la plasticité des lymphocytes T régulateurs et dans le maintien de leur fonction suppressive. Ainsi ce hub de signalisation représente une cible thérapeutique pour augmenter les fonctions des Tregs dans le cadre des maladies inflammatoires ou réduire leurs fonctions suppressives pour favoriser les réponses immunes anti-tumorales dans le cadre du cancer. / Regulatory T cells (Treg) are of paramount importance for restraining excessive immune responses and their manipulation holds enormous therapeutic potential. Our recent results using a congenic rat model suggested that the integrity of Vav1/Themis1 T-cell receptor signaling hub plays a crucial role in Treg suppressive function. Indeed, Themis1 deficiency in BN, but not in LEW rats, led to the development of inflammatory bowel disease (IBD), linked to a defect in Treg suppressive function. Genetic studies revealed that this phenotype depended on a 117 Kb genomic locus, containing the R63W polymorphism on Vav1 that impacted its expression and functions. To test the importance of the Vav1/Themis1 TCR signaling hub in Treg function, we generated Themis1-T-/- mice expressing conditionally Themis1 in thymocytes, but not in peripheral T cells. In contrast to regular germline Themis1 knockout mice, these mice were not lymphopenic and exhibited normal proportions of CD4+ T cells in the thymus and in peripheral lymphoid organs. Next, Themis1-T-/- mice were crossed with Vav1R63W mice to assess the impact of these combined mutations on Treg suppressive functions. Using in vitro approaches, together with in vivo analyses of IBD, we showed that suppressive activity of Treg was impaired in Themis1-deficient mice harboring the mutated Vav1; this defect is linked to higher production of IL-17 and IFNg. Functional studies showed that Themis1-deficient associated with the mutated Vav1 induced a defect in Erk and P65 phosphorylation after TCR engagement. Interestingly, the inhibition of the SHP-1 phosphatase restore the functional defect of Tregs. Together, these data showed that Themis1, Vav1 and SHP-1 cooperate in the signaling hub to regulate the suppressive function of regulatory T cells. Thus, this signaling hub represents a therapeutic target to enhance the suppressive functions of Tregs in the context of autoimmune and inflammatory diseases or to decrease their functions to favor anti-tumoral immune responses.

Silver (Ag) is a precious metal with a low stacking fault energy that is known to form copious nanoscale coherent twin boundaries during magnetron sputtering synthesis. Nanotwinned Ag metals are potentially attractive for creating new interface-dominated nanomaterials with unprecedented mechanical and physical properties. Grain-boundary segregation of solute elements has been found to increase the stability of interfaces and hardness of nanocrystalline metals. However, heavily alloying inevitably complicates the underlying deformation mechanisms due to the hardening effects of solutes, or a change of stacking fault energies in Ag caused by alloying. For the above reasons, we developed a microalloying (or doping) strategy by carefully selecting Cu as the primary impurity – a solute that is predicted to have no solid-solution strengthening effect in Ag when its content is below 3.0 wt.%. Neither will Cu affect the stacking fault energy of Ag at a concentration <1.0 wt.%. Moreover, Cu atoms are ~12% smaller than Ag ones, and Ag-Cu is an immiscible system, which facilitates the segregation of Cu into high-energy interface sites such as grain-boundaries and twin-boundary defects. In this thesis, large-scale hybrid Monte-Carlo and molecular dynamics simulations are used to study the unexplored mechanical behavior of Cu-segregated nanocrystalline nanotwinned Ag. First, the small-scale mechanics of solute Cu segregation and its effects on incipient plasticity mechanisms in nanotwinned Ag were studied. It was found that solute Cu atoms are segregated concurrently to grain boundaries and intrinsic twin-boundary kink-step defects. Low segregated Cu contents (< 1 at.%) are found to substantially increase twin-defect stability, leading to a pronounced rise in yield strength at 300 K. Second, atomistic simulations with a constant grain size of 45 nm and a wide range of twin boundary spacings were performed to investigate the Hall-Petch strength limit in nanocrystalline nanotwinned Ag containing either perfect or kinked twin boundaries. Three distinct strength regions were discovered as twin boundary decreases, delineated by normal Hall-Petch strengthening with a positive slope, the grain-boundary-dictated mechanism with near-zero Hall-Petch slope, and twin-boundary defect induced softening mechanism with a negative Hall-Petch slope. Third, by systematically studying smaller grain sizes, we find that the “strongest” size for pure nanotwinned Ag is achieved for a grain size of ~16 nm, below which softening occurs. The controlling plastic deformation mechanism changes from dislocation nucleation to grain boundary motion. This transition decreases to smaller grain sizes when Cu contents are segregated to the interfaces. Our simulations show that continuous Hall-Petch strengthening without softening, down to grain sizes as small as 6 nm, is reached when adding Cu atoms up to 12 at. %. For Cu contents ≥ 15 at. %, however, the predominant plastic deformation mechanism changes to shear-band induced softening. The present thesis provides new fundamental insights into solute segregation, and strengthening mechanisms mediated by grain boundaries and twin boundaries in face-centered cubic Ag metals, which is expected to motivate experimental studies on new nanotwinned metals with superior mechanical properties controlled by microalloying.

Reef-building corals (Order: Scleractinia) are undergoing rapid taxonomic revision after molecular systematics disputed the relationships at all taxonomic levels within traditional classification. New morphological characters are being used to produce evolutionary relationships supported by molecular phylogenetics. While these characters are providing more congruent taxonomic relationships, their variation has not been fully explored. Additionally, phenotypic plasticity (changes in morphology resulting from environmental factors influencing the expressed phenotype despite a shared genotype) is prevalent amongst Scleractinia. In order to better understand the nature of these characters and explore their variation, I created a series of aquaria-based experiments designed to test the stability of these new morphological characters in response to differing environmental conditions. Light intensity and temperature were chosen as the environmental factors varied in these experiments on the basis of being a known trigger for environmentally-driven plasticity and their importance in calcification rate. In addition to aquaria-based phenotypic plasticity experiments I also examined a group (Family: Euphylliidae) within Scleractinia that had been divided by molecular phylogeny into two disparate groups. My research focused on morphological features viewed at magnifications observable by scanning electron microscopy (SEM) called micromorphology. Although variation in the skeletal micromorphology is observable, the new morphological characters that are used in taxonomy display only small amounts of variation caused by changing environmental conditions and were found to be stable for use in taxonomic studies. Additionally, I found a few micromorphological features distinguishing the two groups previously assigned to Euphylliidae including the shape of the septal margins and the fine-scale skeletal texture.

Motor learning is an important component of daily life: humans are constantly adjusting their movements and acquiring new skills in order to meet the demands of their environment. Motor learning also contributes to neurorehabilitation, so it is therefore important to understand the neural mechanisms underlying motor learning so that these mechanisms can be exploited to promote neurorehabilitation after central nervous system injury. This dissertation focuses on three distinct methods of improving motor learning in healthy adults. In Chapter 2, we tested the effects of perturbation schedule on retention of a locomotor adaptation. The results of this work demonstrated that introducing a perturbation slowly and incrementally versus introducing a perturbation abruptly produces similar behavioral expression of locomotor memories across days. In Chapter 3, we tested whether administering 200 mg of caffeine immediately after practicing a novel motor skill enhances retention of that skill 24 hours later. However, we found that post-practice caffeine administration did not significantly improve retention of the motor skill. In combination with previous reports, these results suggest that the effects of post-practice caffeine administration are likely task-specific. In Chapter 4, we examined the interactions between hand use, practice-dependent plasticity and motor learning. We found that experimentally immobilizing the left hand for 8 hours facilitates subsequent practice-dependent changes in corticospinal excitability in a topographically-specific manner. In contrast, immobilization did not facilitate practice-dependent changes in TMS-evoked thumb movements, nor did it promote learning or retention of a ballistic motor skill. Although it is thought that practice-dependent changes in corticospinal excitability are an important and potentially causal contributor to motor memory, the results of this work indicate that experimentally enhancing practice-dependent changes in corticospinal excitability is not sufficient to promote motor learning. In sum, although none of the experimental interventions tested here substantially improved motor learning, these experiments highlight the influence of various mechanisms on motor learning in the intact nervous system.

My dissertation focused on understanding how different areas of the brain coordinate in networks to drive higher cognitive functions, and how damage, changes the brain’s synchronized activity (or functional connectivity) in the short and long term. In this dissertation, I studied the functional connectivity of brain networks that are thought to underlie emotion and decision-making, and how these networks change in the face of neurological injury. In my first set of experiments, I studied participants with chronic focal brain damage to determine how damage to brain areas which have been identified as important in emotion and decision-making behaviors (amygdala, ventromedial prefrontal cortex, & insula), affected connectivity of brain networks, and how changes in connectivity following damage to these areas related to emotion and decision-making behavior. Supporting my predictions, I found evidence that damage to the amygdala, ventromedial prefrontal cortex, and insula all result in significantly weaker connections between a network of areas important for assigning value to stimuli. Additionally, I found that stronger connectivity in this valuation network was significantly positively associated with performance on ratings of disgusted faces, while stronger connectivity in a network important for processing emotional salience was significantly positively correlated with decision-making performance. In the second set of studies in this dissertation, I utilized a population of epilepsy patients who were undergoing brain surgery to treat their seizures to investigate how a brain network related to emotional salience changed from before to after surgery. This approach allowed me to study how the connectivity and associated behavior of this network changed from preoperative baseline, to the weeks and months after part of this network was removed. While I expected a decline in this network in the weeks following surgery, instead I found a significant positive correlation between preoperative and acute postoperative connectivity in a subset of this network. However, my hypothesis that there would be a significant increase in the connectivity of this network between acute and chronic postoperative epochs was supported. I only have partial evidence for a significant correlation between the change in salience network connectivity between preoperative and acute postoperative assessments and the associated change in decision-making behavior. This correlation was in the opposite direction of my hypothesis, with increased change in connectivity being positively associated with change in risk-taking behavior. I did not observe a significant correlation between the change in network connectivity and change in behavior across acute and chronic measurements. These findings provide important insight on how measures of network connectivity can inform theories of neuroplasticity and reorganization following brain damage. Understanding how these networks change over time, and how changes in these networks relate to behavioral outcomes, are critical for the development and effective deployment of therapeutic interventions. Together, these studies provide a foundation for further study, demonstrating that these networks change over time with damage, and the residual network strength is associated with performance on measures of emotion and decision-making.