<p>PUFAs serve many important biological and physiological functions within the body and are key for the structure and function of the brain. Omega-6 and omega-3 PUFAs are found in abundance in phospholipids of neuronal membranes that impart structure and function of neurons. Omega-6 PUFAs are instrumental for neurotransmission, neuronal elongation, and neuritogenesis; whereas, omega-3 PUFAs promote neuronal maturation through synaptogenesis. The types of PUFAs incorporated into neuronal membranes is especially important in determining the progression of development. The processes of neurogenesis, neuritogenesis and elongation require large amounts of PUFAs to be incorporated into the membrane phospholipids. To accommodate for the high PUFA needs, maternal dietary PUFA, especially EPA and DHA, recommendations, mobilization of fatty acids into maternal circulation increases, and the accretion rate of PUFA are increased. If maternal nutritional inadequacy of PUFAs occurs during gestation, this can result in impaired cognition, behavioral abnormalities, reduced number of neurons, decreased dendric arborization, altered myelin sheath, and a reduction in brain size. </p> <p> Even though the essentiality of PUFAs in neuronal development is widely accepted, the mechanism is not well understood. There is a lack of consensus in the current literature on the effects of individual PUFAs on each stage of neuronal development and the molecular pathways involved. Despite the inconsistent evidence, the results of numerous studies have consistently suggested that neuronal membrane PUFA composition is associated with neuronal development outcomes, such as number of neurons and neurites, neurite length, and neurotransmitter release. The varying results may be the result of methodological discrepancies with PUFA composition and concentrations, as well as the models used for neuronal development. Additionally, very few studies have taken into consideration the competitive relationship of omega-6 and omega-3 PUFAs in the body when assessing neurodevelopment. </p> <p> This thesis was focused on addressing the role of PUFAs in neuronal development and to address some of the inconsistencies in the literature. attempt to elucidate the individual roles of ALA, ARA, and EPA on neuronal membrane composition and neuronal development. The aim of the thesis research project was to assess the impact of individual PUFAs on neuronal membrane PUFA composition, the membrane n-6:n-3 ratio, and the morphology of SH-SY5Y cells during differentiation. The results of this study demonstrated that supplementation of individual PUFAs alters membrane PUFA composition and the n-6:n-3 ratio. However, there wasn’t a significant effect on neurite number with ALA, ARA, and EPA treatment. Lastly, ARA treatment decreased cell viability compared to the other treatments and the BSA control. Furthermore, additional research needs to be conducted to address other morphological measures and functional outcomes, such as neurotransmitter production and release.</p>

Peripheral nerves have demonstrated the ability to bridge gaps of up to 6 mm. Peripheral Nerve System injury sites beyond this range need autograft or allograft surgery. Central Nerve System cells do not allow spontaneous regeneration due to the intrinsic environmental inhibition. Although stem cell therapy seems to be a promising approach towards nerve repair, it is essential to use the distinct three-dimensional architecture of a cell scaffold with proper biomolecule embedding in order to ensure that the local environment can be controlled well enough for growth and survival. Many approaches have been developed for the fabrication of 3D scaffolds, and more recently, fiber-based scaffolds produced via the electrospinning have been garnering increasing interest, as it offers the opportunity for control over fiber composition, as well as fiber mesh porosity using a relatively simple experimental setup. All these attributes make electrospun fibers a new class of promising scaffolds for neural tissue engineering. Therefore, the purpose of this doctoral study is to investigate the use of the novel material PGD and its derivative PGDF for obtaining fiber scaffolds using the electrospinning. The performance of these scaffolds, combined with neural lineage cells derived from ESCs, was evaluated by the dissolvability test, Raman spectroscopy, cell viability assay, real time PCR, Immunocytochemistry, extracellular electrophysiology, etc. The newly designed collector makes it possible to easily obtain fibers with adequate length and integrity. The utilization of a solvent like ethanol and water for electrospinning of fibrous scaffolds provides a potentially less toxic and more biocompatible fabrication method. Cell viability testing demonstrated that the addition of gelatin leads to significant improvement of cell proliferation on the scaffolds. Both real time PCR and Immunocytochemistry analysis indicated that motor neuron differentiation was achieved through the high motor neuron gene expression using the metabolites approach. The addition of Fumaric acid into fiber scaffolds further promoted the differentiation. Based on the results, this newly fabricated electrospun fiber scaffold, combined with neural lineage cells, provides a potential alternate strategy for nerve injury repair.

ROS are frequently associated with deleterious effects caused by oxidative stress. Despite the harmful effects of non-specific oxidation, ROS also function as signal transduction molecules that regulate various biological processes, including stem cell proliferation and differentiation. Here we show that mitochondrial ROS level determines cell fate during differentiation of the pluripotent stem cell line P19. As stem cells in general, P19 cells are characterized by a low respiration activity, accompanied by a low level of ROS formation. Nevertheless, we found that P19 cells contain fully assembled mitochondrial electron transport chain supercomplexes (respirasomes), suggesting that low respiration activity may serve as a protective mechanism against ROS. Upon elevated mitochondrial ROS formation, the proliferative potential of P19 cells is decreased due to longer S phase of the cell cycle. Our data show that besides being harmful, mitochondrial ROS production regulates the differentiation potential of P19 cells: elevated mitochondrial ROS level favours trophoblast differentiation, whereas preventing neuron differentiation. Therefore, our results suggest that mitochondrial ROS level serves as an important factor that directs differentiation towards certain cell types while preventing others.