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Regenerative medicine offers tremendous potential for the treatment of irreversible damage to the brain. Activation of quiescent adult neural stem cells by clinical means to regenerate tissue can improve pathological outcomes of patients afflicted by brain trauma. Control of the cell- cycle is important in activating quiescent neural stem cells for the purpose of enhancing adult neurogenesis. Here, we uncover the role of cell-cycle regulatory transcription factors E2F1 and E2F3 in adult neural stem cell activation and characterize it. We hypothesize that the Retinoblastoma-E2F pathway is crucial for neural stem cell activation. We characterized the requirement of E2Fs1/3 for adult neural stem cells activation through a combination of multiple knockout timepoints in mice and novel markers used to identify distinct neural stem cell sub- populations. The results show a marked reduction in the neurogenic capacity of the adult brain, with common markers of proliferation and different progenitor-cell lineages decreased. Additionally, the ability of quiescent neural stem cells to transition to an active state is reduced. A whole genome-analysis of RNA isolated from E2Fs1/3-knockout adult neural stem cells has shown a shift from an active identity-state to a quiescent one. In the future, E2Fs1/3 could emerge as key regulators of quiescent stem cell activation, and thus could be potential targets for therapeutic control in order to enhance neurogenesis in patients with brain pathology.

Bioengineered skin is a promising treatment for chronic skin wounds because of its ability to promptly promote wound healing at the injury site and to restore the skin’s epidermal and dermal structures and functions. Despite some level of clinical success, commercially available bioengineered skin substitutes are still limited by a high incidence of infection, a lack of mechanical integrity, and a slow rate of tissue ingrowth from the surrounding wound margin. To address these challenges, we propose to engineer novel polymer composite matrices for dermal regeneration. These matrices consist of two different electrospun polymer layers which create a composite matrix made up of a highly porous three-dimensional fibrous network. Each composite matrix contains a biodegradable electrospun “dermal” layer which acts as a scaffold for dermal cell ingrowth and tissue regeneration and a non-degradable electrospun “epidermal” layer that serves as a provisional barrier to protect the wound from environmental insult. To evaluate the success of our designs, we performed quantitative analyses of the physical properties of our electrospun scaffolds including fiber diameter and angle analyses and mechanical properties. We found our electrospun scaffolds are comprised of a random network of fibers ranging from approximately 0.2 – 5µm in diameter. They exhibit several mechanical properties that are similar to those measured in native skin tissue, including tangent elastic modulus and strain at failure. We have also found the proposed nanofibrous scaffolds to be capable of supporting normal human fibroblast attachment and migration. Our scaffolds show similar attachment to tissue culture polystyrene controls and better attachment than collagen-GAG sponge controls. The dermal layer of our scaffolds show fibroblast outgrowth rates between 185 - 206µm/day, which is similar to rates observed by others in collagen-GAG sponges and wounds. The promising findings from these in vitro studies warrant that our novel electrospun dermal regeneration matrix be further developed.

Bioengineered skin is a promising treatment for chronic skin wounds because of its ability to promptly promote wound healing at the injury site and to restore the skin’s epidermal and dermal structures and functions. Despite some level of clinical success, commercially available bioengineered skin substitutes are still limited by a high incidence of infection, a lack of mechanical integrity, and a slow rate of tissue ingrowth from the surrounding wound margin. To address these challenges, we propose to engineer novel polymer composite matrices for dermal regeneration. These matrices consist of two different electrospun polymer layers which create a composite matrix made up of a highly porous three-dimensional fibrous network. Each composite matrix contains a biodegradable electrospun “dermal” layer which acts as a scaffold for dermal cell ingrowth and tissue regeneration and a non-degradable electrospun “epidermal” layer that serves as a provisional barrier to protect the wound from environmental insult. To evaluate the success of our designs, we performed quantitative analyses of the physical properties of our electrospun scaffolds including fiber diameter and angle analyses and mechanical properties. We found our electrospun scaffolds are comprised of a random network of fibers ranging from approximately 0.2 – 5µm in diameter. They exhibit several mechanical properties that are similar to those measured in native skin tissue, including tangent elastic modulus and strain at failure. We have also found the proposed nanofibrous scaffolds to be capable of supporting normal human fibroblast attachment and migration. Our scaffolds show similar attachment to tissue culture polystyrene controls and better attachment than collagen-GAG sponge controls. The dermal layer of our scaffolds show fibroblast outgrowth rates between 185 - 206µm/day, which is similar to rates observed by others in collagen-GAG sponges and wounds. The promising findings from these in vitro studies warrant that our novel electrospun dermal regeneration matrix be further developed.

In the light of increasing spectral congestion in the land mobile radio bands below 1 GHz,a renewed interest in Single Sideband (SSB) modulation has been observed world-wide. In this context, much of the work described in this thesis has been directed towards assessing and developing audio signal processing techniques that enable the spectrum and power efficient SSB systems to acheive a comparable performance with that of existing wideband modulation systems. Statistical properties of the land mobile-radio propagation are presented showing that the received signal can be adversely affected by unwanted random phase and envelope modulations. Their effect upon AM, FM and SSB systems are discussed. A novel space diversity system employing a technique termed as Feedforward Signal Regeneration (FFSR) is presented which simultaneously performs the operation of an equal gain combining and suppression of both the random envelope and phase modulations. In connection with the use of FFSR techniques in SSB systems, a phase-locked Transparent-Tone-In-Band (TTIB) pilot-tone configuration which allows the transmission of a "transparent" pilot-tone, positioned centrally within the audio bandwidth, is discussed. Finally, efficient coherent and non-coherent data transmission in the mobile-radio environment is shown to be possible by the use of the TTIB/FFSR diversity system.

"Engineered muscle tissue offers a promising solution for the treatment of large muscle defects. Three-dimensional tissue engineered matrices, such as microthreads, can be used to grow new myofibers that will reduce scar formation and integrate easily into native myofibers. We hypothesize that adsorbing growth factors to the surface of braided collagen scaffolds using crosslinking strategies will promote muscle derived fibroblastic cell (MDFC) attachment and growth, which will serve as a platform for delivering cells to large muscle defects for muscle regeneration. To test this hypothesis, self-assembled type I collagen threads were braided and crosslinked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) with and without heparin and 5 ng/mL, 10 ng/mL, or 50 ng/mL fibroblast growth factor (FGF-2) bound to the surface. Using immunhistochemistry, braided collagen scaffolds showed the presence of FGF-2 on the surface, and braiding the microthreads increased the mechanical properties compared to single threads. To determine the effect of FGF-2 on MDFC attachment, growth, and alignment, scaffolds were seeded with a MDFC cell suspension for 4 hours using a PDMS mold with a sealed 1 mm by 12 mm channel and cultured for 1, 5, or 7 days. After 1 day of culture, the results show a significant increase in cell attachment on braids crosslinked with EDC/NHS with heparin and no significant difference in attachment between the different concentrations of FGF-2 and EDC/NHS crosslinked scaffolds. After 7 days in culture, the MDFCs responded to FGF-2 with a positive linear correlation between growth rate and concentration of FGF-2 on the surface. Additionally, all control scaffolds showed cellular alignment after 7 days, while MDFCs on FGF-2 modified scaffolds showed limited alignment. These results show braided collagen scaffolds crosslinked with EDC/NHS with heparin delivering a controlled quantity of FGF-2 can support MDFC attachment and growth, which may serve as an exciting new approach to facilitate the growth and ultimately the delivery of cells to large defects in muscle regeneration."

Digitized by Kansas Correctional Industries

Chronic Liver Disease (CLD) and Acute Liver Failure (ALF) are serious medical syndromes. Current therapeutic options consist of managing complications, and liver transplant. Even if liver transplantation is thought to be suitable for CLD or ALF patients, there are not enough organs available and thus increasingly more deaths occur on the transplant waiting list. Therefore, there is a pressing need to develop additional therapies. This thesis firstly systematically reviews trials in autologous cell therapies as possible treatments for patients with cirrhosis. The published literature is imperfect and the difference in trial design means it has not been possible to conduct a meta-analysis. Regardless of these shortcomings, cell therapy is a potentially positive prospect. In ALF and CLD monocyte-macrophages have diverse roles within the liver. Monocyte and immune cell changes in ALF are investigated and it is demonstrated for the first time that patients with paracetamol induced ALF have a significantly altered blood compartment and that these changes correlate with patient outcome. It is possible that these results may help stratify which patients may spontaneously survive and which patients may require an emergency liver transplant. Furthermore, modulation of these changes may improve outcomes for patients. The thesis also examines monocyte-macrophages in cirrhotic patients and demonstrates the feasibility of differentiating cirrhotic patients’ monocytes into functional macrophages, comparable to healthy volunteers in a Good Manufacturing Practice (GMP) environment. A first in-man trial using macrophages infused to patients with cirrhosis as a potential new treatment is also detailed. Finally, this thesis outlines developmental work for cell therapy in patients with cirrhosis in the multi-centre REALISTIC trial. Patients were randomly assigned to receive; standard medical care, Granulocyte Stimulating Factor (GCSF) injections alone or GCSF combined with repeated stem cell infusion.

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 (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.