The role of semaphorins in response to injury in C. elegans neurons

When neural tissue is injured by trauma, delicate neuronal processes such as axons and dendrites are prone to lesion damage and often disconnect. The molecular, cellular, and circuit mechanisms that underlie the regrowth and reconnection of these processes and the recovery of behavior are major challenges in the fields of neuroscience, regeneration, and resilience. At the molecular and cellular levels, signaling pathways that mediate neuronal growth cone guidance during development can play a role in neuronal regeneration and recovery from injury. One family of signaling proteins involved in this process comprises the highly conserved semaphorins and their receptors, the plexins. Across various species, from C. elegans to humans, semaphorins and plexins are crucial for axon pathfinding and synapse formation during development. In the mammalian nervous system, the semaphorin signaling system is comprised of more than 20 semaphorins and 9 plexins, whereas the C. elegans genome only encodes 3 semaphorins and 2 plexin receptors. Among them, the transmembrane semaphorins, SMP-1 and SMP-2, signal through their receptor PLX-1, while the secreted semaphorin MAB-20, signals through PLX-2. This dissertation explores the role of semaphorin signaling in neuroregeneration in vivo, by making use of the experimental advantages of Caenorhabditis elegans. Importantly, this versatile model animal has the natural ability to regenerate neuronal processes after injury and optic methods were developed to precisely disconnect single neurites in otherwise intact animals using laser microsurgery. Moreover, the semaphorin system is relatively simple and genetically amenable, and transgenic, microscopy and behavior analysis methods are well established. The development and assessment of a new laser microsurgery system as part of this thesis allowed reliable and accurate disconnection of identifiable axons and dendrites. The elucidated expression patterns and involvement of C. elegans semaphorins in neural regeneration have shed significant light with regard to the role this pathway plays in C. elegans regeneration and added to the field of knowledge of neural regeneration research. The findings reveal that regrowth and reconnection are more prevalent in the absence of both plexin receptors and the secreted semaphorin MAB-20. This suggests that the semaphorin signaling in this system restricts neural growth, possibly to prevent aberrant reconnection. The membrane-bound SMP-1 and SMP-2 might have a redundant role, signaling through PLX-1. These results align with the inhibitory effects of semaphorin signaling on axonal growth and guidance during development in the mammalian system. Therefore, secreted and membrane-bound semaphorin signaling pathways restrict regeneration using distinct processes, likely involving spatial specificity and recurrent signals. Findings such as the ones presented in this thesis delve deeper into the mechanisms and factors involved in promoting regeneration and aid to uncover valuable insights that could assist in overcoming the challenges faced by regenerative medicine in treating central nervous system injuries and disorders

A Study of Infrared Femtosecond Laser Irradiation on Monolayer Graphene on SiO2/Si Substrate

Graphene is a single hexagonal atomic carbon layer. Since its discovery, graphene is emerging as an exciting and promising new material to impact various areas of fundamental research and technology. It has potentially useful electrical properties for device applications such as graphene photodetectors and graphene-based sensors. This thesis focuses on the femtosecond laser processing of graphene from both scientific and industrial points of view. Started from the manufacturing process, a new manufacturing route for graphene devices based on a femtosecond laser system is explored. In this thesis, the graphene ablation threshold was determined in the range of 100 mJ/cm2. In this deposited fluence range, selective removal of graphene was achieved using femtosecond laser processing with little damage to the SiO2 /Si substrate. This finding supports the feasibility of direct patterning of graphene for silicon-substrate field effect transistors (FETs) as the gate dielectric, silicon dioxide is only negligibly removed (2~10 nm) and no damage occurs to the silicon. Beyond the selective removal of graphene, the effects of exposing femtosecond laser pulses on a monolayer of graphene deposited on a SiO2/Si substrate is also studied under subthreshold irradiation conditions. It has been demonstrated that a femtosecond laser can induce defects on exposure. The dependence of the D, G, and 2D Raman spectrum lines on various laser pulse energies was evaluated using Raman Spectroscopy. The I (D)/I (G) ratio was seen to increase with increasing laser energy. The increase in the D’ (intravalley phonon and defect scattering) peak at 1620 cm-1 appeared as defective graphene. These findings provide an opportunity for tuning graphene properties locally by applying femtosecond laser pulses. Applications might include p-n junctions, and the graphene doping process. To explore the power absorption process in graphene and the SiO2/Si substrate, a theoretical model was developed based on the transfer-matrix method. The results revealed that the most significant absorption was in the silicon substrate. The light reflection form each layer was considered. The model shows the temperature oscillations are more significant in the silicon layer compared to the silicon dioxide which can provide a theoretical rationale for the swelling effect observed in the experiments. This model can assist in the choice of laser parameters chosen for future laser systems used in the production of graphene devices

DEVELOPMENT OF INNOVATIVE MULTICOMPARTMENT MICROFLUIDIC PLATFORMS TO INVESTIGATE TRAUMATIC AXONAL INJURY

Compartmentalization of cell body from the axon of a neuron is an important aspect in studying the influence of microenvironments. Microenvironment is an integral part of neuronal studies involving traumatic axonal injuries (TAI). While TAI is one of the possible outcomes of various forms of traumatic insults to the central nervous system (CNS) and peripheral nervous system (PNS), many of the mechanistic details are still unknown, it can be agreed that the level of injury often dictates the functional deficit. This motivates the question, what is occurring at both the morphological and biomolecular scale in CNS and PNS axons during and throughout the recovery phase after injury? And, are there any treatment strategies that could be applied to improve the recovery and regeneration of the axons subject to TAI? Motivated by this, I propose to develop novel microfluidic platforms as a part of my master’s thesis to allow unprecedented, physiologically relevant focal and graded mechanical injury and observation to both CNS and PNS axons. My research for this thesis can be broadly classified into two fold. 1) I examined the regenerative effects of the members of the Glial cell line-derived neurotrophic factor (GDNF), a family of neurotrophic factors after axotomy. This work resulted in the discovery of the fact that GDNF is the most potent neurotrophic factor among the family of growth factors for axon regeneration in dorsal root ganglion (DRG) neurons after in vitro axotomy. It was also found that GDNF locally applied to cell body better promotes axonal regeneration in comparison to when applied locally to axons. 2) Development and refinement of existing axon injury microplatform (AIM) to closely mimic physiological conditions during traumatic injury in CNS neurons. This work was my attempt in improving already existing microfluidic compression platform. I successfully developed a displacement control injury device and demonstrated displacement control as a proof of principle. Further development of these microfluidic platforms will significantly contribute to the field of basic neuroscience, neurobiology, and biomedical engineering

Étude expérimentale de procédés de bioimpression assistés par laser femtoseconde

This manuscript deals with the experimental study of two bioprinting processes assisted by femtosecond laser at a wavelength of 1030 nm. Indeed, femtosecond lasers are an interesting choice for bioprinting: the high versatility of materials which can be deposited and the negligible heat affected zone are advantages to print complex biological structures without compromising viability and functionality of the transferred biological materials. Firstly, femtosecond laser assisted bioprinting with a metallic absorbing layer was studied on a bioprinter adapted for cell printing (MODULAB®). An experimental study was conducted, observing the laser induced jet of liquid with a time-resolved imaging system (TRI) and printing on receiver substrates (cell culture well plate). The bioink rheology, some laser parameters, and the laser focus position were changed during the experiments. Cell viability assays after the printing enabled to identify an optimal energy of 3 μJ. The study of the laser focus position variation allowed predicting the tolerance range of the laser focus position: for 3.5 μJ and an equivalent numerical aperture (NA) of 0.125, the maximum tolerance in the “z” direction was of 60 μm in order to print. Secondly, femtosecond laser assisted bioprinting without an absorbing layer was studied on an experimental set-up comprising a reservoir of bioink. Some key operating parameters were studied (focalization position, NA of the focalization objective, printed drop diameter, printing jet height by TRI, maximum transfer distance for printing). The printing was reproducible for a printing distance from 75 % hmax to 100 % hmax, with hmax corresponding to the maximum printing jet height for a given experimental condition. Using the reservoir of bioink enabled to find a tolerant focalization position z: Δz was calculated (Zernike polynomial and the spherical aberration) and measured. Experimentally, Δz ranged from 0 to 60 μm depending on the bioink and the NA. It was maximal at NA 0.4. This tolerance is high compared to the depth of field in the air (4 μm at NA 0.4) but low compared to the tolerance of the receiver substrate position which can vary to 25 % hmax according to the reproducibility range.Ce mémoire est consacré à l’étude expérimentale de deux procédés de bioimpression assistée par laser femtoseconde, fonctionnant à 1030 nm. En effet, les lasers femtosecondes constituent un choix intéressant pour la bioimpression: la versatilité des matériaux qui peuvent être déposés et la zone affectée thermiquement négligeable sont des atouts pour l’impression de structures biologiques complexes, sans compromettre la viabilité et la fonctionnalité des matériaux biologiques transférés. Tout d’abord, la bioimpression assistée par laser femtoseconde avec couche absorbante métallique a été étudiée sur une bioimprimante adaptée au transfert de cellules (MODULAB®). Une étude expérimentale a été menée par observation du jet induit par laser grâce à un système d’imagerie résolue en temps (TRI) et par impression sur receveur (puits de culture). La rhéologie de la bioencre, certains paramètres laser, ainsi que la position de focalisation laser ont été variés lors des expériences. Des tests de viabilité cellulaire après l’impression ont permis d’identifier une énergie optimale de 3 μJ. L’étude de la variation de la position de focalisation a permis de prédire la plage de tolérance de la position de focalisation du laser : pour une énergie de 3,5 μJ et une ON équivalente de 0,125, la tolérance maximale dans la direction « z » était de 60 μm pour pouvoir imprimer.Dans un second temps, la bioimpression assistée par laser femtoseconde sans couche absorbante a été étudiée sur un montage expérimental comprenant un réservoir de bioencre via des paramètres opératoires clés (position de focalisation, ouverture numérique de l’objectif de focalisation, diamètre de goutte imprimé, la hauteur du jet de l’impression par TRI, la distance de transfert limite pour imprimer). L’impression était reproductible pour une distance d’impression de 75 % hmax à 100 % hmax, hmax étant la hauteur maximale du jet d’impression pour une condition expérimentale. L’utilisation du réservoir de bioencre a permis de trouver une position de focalisation z tolérante: Δz a été calculée (Zernike et l’aberration sphérique) et mesurée. Expérimentalement, Δz valait de 0 à 60 μm selon la bioencre et l’ON. Elle était maximale à l’ON 0,4. Cette tolérance est grande devant la profondeur de champs dans l’air (4 μm à l’ON 0,4) mais faible au regard de la tolérance sur la position du receveur qui peut subir une variation de 25% hmax, d’après la plage de reproductibilité