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This thesis reports three projects on mussel-inspired biomimetic materials based on dopamine crosslinkers. First, polyethylene glycol diacrylate (PEGDA) hydrogels with excellent cell attachment and tunable stiffness was fabricated based on this novel crosslinker. In vitro tests proved that the designed hydrogels had excellent cell adhesion, suggesting the developed hydrogels are promising for applications in tissue engineering. Second, dopamine crosslinker-conjugated gelatin-polycparolactone (PCL) nanofibrous sheet was developed. The sheet was then employed successfully to treat stomach incision without suture during surgery, showing promising to deal with treatments of fragile tissues and to avoid suture caused stress concentration. Third, a facile strategy to wrap cell into a tissue-engineered scaffold was developed, which is a self-rolling and conductive dopamine-based film. The RTPCR test indicated that cells have higher level of differentiation with higher concentration of MWCNTs. This suggests that the self-rolling conductive WCNT-dopamine-PEG hydrogel is a promising scaffolding material for bone regeneration. / October 2015

The zebrafish has an amazing capacity for regeneration which includes regeneration of neurons within the central nervous system (CNS) both during development and into adulthood. This attribute makes the zebrafish a valuable tool in the study of regeneration. In this thesis, the research focussed on the regeneration of a specific type of cell in the CNS, dopaminergic (DA) neurons. The DA system of the zebrafish is believed to be evolutionarily conserved with comparable DA populations found in the brain of mammals. Dissimilar to mammals, however, the zebrafish is capable of regenerating various types of neurons and their axons. Thus, the zebrafish DA system provides an excellent model to study replacement of this specific and important cell type in the adult CNS. We have developed a novel toxin ablation paradigm to specifically ablate select groups of DA neurons in the adult zebrafish diencephalon, leaving other DA populations unaffected. To do this a selective DA toxin, 6-hydroxydopamine, was used. One of the ablated DA diencephalic populations is the only source of dopaminergic spinal innervation in the zebrafish. Their ablation leads to a loss of DA spinal axons following our toxin ablation. The ascending projection of the diencephalic population ablated by the toxin has been suggested as the most likely candidate for a zebrafish equivalent of the mammalian nigro-striatal pathway. The loss of cells is very specific and reproducible, indicating that these cells are particularly vulnerable to the toxin. Quantification of affected populations at various time-points post ablation was carried out to determine the capacity for regeneration of DA neurons in the CNS of zebrafish. This revealed that in some populations neuron numbers returned to those seen in controls. However, in other populations neuron numbers only partially recovered even at late time points. We have shown that this recovery is due to neurogenesis; furthermore, by inducing inflammation after the toxin treatment the recovery of DA cell numbers was accelerated by 50%. Regenerated cells originated from Olig2 positive ependymo-radial glial cells found bordering the diencephalic ventricle. We aimed to investigate the function of this group of ablated neurons through a battery of behavioural tests. These tests revealed deficits in the toxin treated animals’ fine movement, such as is necessary for maintaining shoal cohesion and breeding behaviours, whereas general movement behaviours were not found to be impaired. Zebrafish embryos also present as a great resource in the screening of drugs. Their fast and well characterised early development makes them an ideal tool for investigating previously untested neuroprotectants. A reproducible ablation paradigm similar to that established in the adults was also established in the zebrafish embryo. This was then used as a tool to investigate potential novel neuroprotectants. This screen revealed two new flavonoid compounds which had the ability to induce full protection of the affected dopaminergic cells in the zebrafish embryonic brain. The embryonic ablation model therefore represents a vertebrate in vivo model system for future high throughput screening of neuroprotective compounds against toxin induced DA cell loss. Ultimately, understanding how zebrafish functionally regenerate dopaminergic neurons using this ablation model will likely provide a useful tool into the research of neurodegenerative diseases, such as PD.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on December 20, 2007) Includes bibliographical references.

Thesis (Ph. D.)--Illinois State University, 2005. / Title from title page screen, viewed on April 22, 2007. Dissertation Committee: Maarten E.A. Reith (chair), Hou Tak Cheung, Stephen M. Lasley, Robert L. Preston, Brian J. Wilkinson. Includes bibliographical references and abstract. Also available in print.

The short form of dopamine D2 receptor (D2S) functions as a presynaptic autoreceptor on dopamine neurons and has an inhibitory effect on dopaminergic tone. D2-MAPKs pathway is involved in many physiological events like production of prolactin and tyrosine hydroxylase (TH) expression. However, the effect of D2S receptor signalling on MAPKs is cell type specific, and is not fully understood.A recent study in our lab has identified a Gαi-interacting ras-MAPK inhibitor RASA3. Here, we showed that RASA3 is the key effector in D2-induced inhibition of MAPK by knockdown of endogenous RASA3 in the GH4 cell using RASA3 siRNA. We have also transfected a dominant negative RASA3 to compete with the endogenous RASA3 for the binding site on Ras. Both RASA3-siRNA and dominant negative RASA3 blocked D2S-induced inhibition of MAPK activation, clearly implicating that RASA3 is a key effector in Gαi3-dependent D2S mediated MAPKs inhibition To determine whether RASA3’s inhibitory effect could be reconstituted in fibroblast cells, the effect of RASA3 on D2-mediated ERK1/2 activation in COS7 cells was tested. Our results show that both active Gαi2 (or Gαi3) and active RASA3 are required for optimal inhibition of ERK1/2 activation in fibroblast COS7 cells.

Reproductive success in the male rat depends on the ability to recognize appropriate sexual cues, motivation to respond to those cues, and coordination of the necessary motor sequences required to optimize sexual performance and an ejaculatory response. Early maternal environment is important in the normal development of copulatory behavior. Manipulation of this early social stimulation results in alterations in male sexual behavior and in the functioning of mediating endocrine and neurotransmitter systems. The present series of studies were designed to explore the effects of early life maternal deprivation and replacement maternal licking-like stimulation on the development of male rat sexual behavior and the neurophysiological mechanisms which mediate sexual performance with specific attention to the dopamine (DA) and androgen systems. Long-Evans male rats were reared with or without their mothers through the use of the artificial rearing (AR) paradigm. Half of the AR rats were provided with licking-like stimulation, consisting of periodic stroking with a paintbrush. In study 1, AR and maternally-reared (MR) rats were tested in adulthood for sexual behavior. Neuronal activation in response to copulation was assessed using an antibody against the protein product of the immediate early gene c-fos in brain regions that sub-serve sexual behavior. Study 2 explored whether sexual behavioral deficits observed in AR males can be reversed by later sexual experience. In this study, animals were sacrificed following a ninth copulatory trial and Fos immunoreactivity, androgen and estrogen-α receptors were assessed. In study 3, the effects of early maternal deprivation on partner preference in both males that are differentially reared, and, female preference towards these males were investigated. This explored if any behavioral deficits observed in AR males could be attributed to differences in their attractivity to females. Study 4 investigated the effects of early maternal deprivation on androgen sensitivity in adult males. Copulatory response to a receptive female was examined post-castration in AR and MR males and again following testosterone replacement. In study 5, levels of extracellular DA were investigated in the nucleus accumbens, an area critical in motivation, prior to and during copulation in sexually experienced AR and MR males using Microdialysis.

Reproductive success in the male rat depends on the ability to recognize appropriate sexual cues, motivation to respond to those cues, and coordination of the necessary motor sequences required to optimize sexual performance and an ejaculatory response. Early maternal environment is important in the normal development of copulatory behavior. Manipulation of this early social stimulation results in alterations in male sexual behavior and in the functioning of mediating endocrine and neurotransmitter systems. The present series of studies were designed to explore the effects of early life maternal deprivation and replacement maternal licking-like stimulation on the development of male rat sexual behavior and the neurophysiological mechanisms which mediate sexual performance with specific attention to the dopamine (DA) and androgen systems. Long-Evans male rats were reared with or without their mothers through the use of the artificial rearing (AR) paradigm. Half of the AR rats were provided with licking-like stimulation, consisting of periodic stroking with a paintbrush. In study 1, AR and maternally-reared (MR) rats were tested in adulthood for sexual behavior. Neuronal activation in response to copulation was assessed using an antibody against the protein product of the immediate early gene c-fos in brain regions that sub-serve sexual behavior. Study 2 explored whether sexual behavioral deficits observed in AR males can be reversed by later sexual experience. In this study, animals were sacrificed following a ninth copulatory trial and Fos immunoreactivity, androgen and estrogen-α receptors were assessed. In study 3, the effects of early maternal deprivation on partner preference in both males that are differentially reared, and, female preference towards these males were investigated. This explored if any behavioral deficits observed in AR males could be attributed to differences in their attractivity to females. Study 4 investigated the effects of early maternal deprivation on androgen sensitivity in adult males. Copulatory response to a receptive female was examined post-castration in AR and MR males and again following testosterone replacement. In study 5, levels of extracellular DA were investigated in the nucleus accumbens, an area critical in motivation, prior to and during copulation in sexually experienced AR and MR males using Microdialysis.