The Effect of Manipulation of the Phosphoinositide 3-kinase Pathway on Axon Regeneration in vivo

Millions worldwide are visually impaired by optic nerve diseases, and glaucoma, the leading cause of irreversible blindness, is due to affect approximately 80 million people by 2020. The optic nerve is comprised of retinal ganglion cell (RGC) axons, and like all mammalian central nervous system axons, they fail to regenerate due to a combination of extrinsic and intrinsic factors. Current treatments have limited success preventing disease progression, and this failed regeneration presents a major barrier to restoring vision. Previous research has demonstrated that manipulation of the phosphoinositide 3-kinase (PI3K) pathway by transgenic knockout of phosphatase and tensin homolog (PTEN) promotes survival and regeneration of murine RGC axons in an optic nerve crush (ONC) model. However, translation issues are yet to be addressed. PI3K converts phosphatidylinositol (3,4)-bis-phosphate (PIP2) lipids to phosphatidylinositol (3,4,5)-tris-phosphate (PIP3). PTEN acts as a pathway regulator, converting PIP3 back into PIP2. PIP3 is a second messenger molecule for several pathways, including the mammalian target of rapamycin (mTOR) pathway, which has been demonstrated to promote axon regeneration in RGCs, the spinal cord, and cortical neurons. The PI3K pathway is activated by growth factors, integrins and cytokine receptors. Integrins have also been shown to promote axon regeneration via the focal adhesion kinase pathway. This thesis aimed to determine the effects on axon regeneration of manipulating the PI3K pathway in various ways. A transgenic approach was adopted to conditionally express the hyperactive p110$\alpha$ and p110$\delta$ PI3K isoforms using viral Cre recombination. Both survival and axon regeneration were significantly increased 4 weeks post-ONC injury in young and aged mice. No significant difference between the two isoforms was observed, except that p110$\alpha$ promoted RGC survival in aged mice whereas p110$\delta$ had no significant effect. This was developed further using viral vectors to upregulate PI3K and to knockdown PTEN for a more translational approach: AAV2.shPTEN.GFP and AAV2.PI3K(p110$\delta$). PTEN knockdown promoted both RGC survival and axon regeneration, while PI3K upregulation promoted axon regeneration. No significant difference in axon regeneration was seen between the two strategies. Where regeneration was seen, activation of the mTOR pathway was demonstrated. It is becoming increasingly clear that strategies need to be combined to achieve long-distance, robust regeneration. In this thesis, PI3K upregulation was combined with PTEN knockdown and then with integrin activation using AAV2.Integrin.V5 and AAV2.Kindlin.GFP viruses. While PI3K upregulation and PTEN knockdown promoted RGC survival and axon regeneration compared to control, this was not significantly more than PI3K upregulation alone. The experiments involving integrin activation were unsuccessful and the viruses need to be investigated further. While the field has achieved small numbers of regenerating axons up to the optic chiasm, key challenges for future work are reaching central targets in the brain and assessing axon regeneration in an aged model to better reflect the clinical setting, where neurodegenerative diseases predominately affect the aging population. In summary, the work in this thesis investigated the pro-regenerative effects of potential gene therapy targets, advancing our knowledge for developing future clinical strategies.I am grateful for the funding I received to complete this PhD. My MRC and Sackler PhD scholarship was funded by the UK Medical Research Council and the Sackler fund. In addition, the work in this thesis was also funded by several grants written by Dr Amanda Barber and Prof Keith R Martin: Wellcome Trust, Fight for Sight (The Eye Research Charity), Cambridge Eye Trust, International Glaucoma Association, and the National Eye Research Centre

Mapping Light Pollution at Utah State University

One of the beauties of modern civilization is seeing the city lighting at night. It provides a feeling of security and is indicative of the power and endeavors of humanity, but over-lighting is a form of pollution. Many outdoor light fixtures spread light in all directions, sending a majority of the light into the sky, away from where we want the light to be on the ground. This light spreading upward is not only wasted light, but it is wasted energy and money, destroys our ability to view the night sky, and has profound effects on nocturnal creatures. The direct harm to certain species then affects the ecosystem it interfaces with, causing damage on a much larger scale than expected. Small changes in lighting practices would positively affect energy use and economies, ecosystems, and the efficiency of astronomical endeavors. This project analyzes the light pollution on Utah State University campus and what can be done with collaboration of the collegiate administration to amend the lighting in Logan

Damaging Economies and Ecosystems: Light Pollution in Logan City

Though the view of city lights is considered a modern beauty, indicative of the strength of the human intellect, it is the cause of an assortment of negative effects. Most street lights emit light radially, sending large portions of the light into the sky, leaving the ground dim. The excess light- light pollution- directed at the atmosphere destroys our ability to view beauty of the night sky and represents a loss of energy and money. Damage is also done to nocturnal and migratory animals as well as greatly affecting local ecosystems. To improve the efficiency of astronomical endeavors, the economy, and ecosystems, small innovations must be made to outdoor light fixtures. This project analyzes the luminosity output of outdoor light fixtures and the ambient light pollution of Utah State University Campus. As Utah State University has a sustainability program, Blue Goes Green, that has already implement lighting codes, the goal of this project is the work with the collegiate administration to renovate old lighting fixtures

Characterizing and Quantifying Night Sky Brightness in Utah and Arizona

As part of a Research Experience for Undergraduates (REU) program with the National Optical Astronomy Observatory (NOAO), I (with mentor Dr. Constance Walker of NOAO) characterized light pollution in and near Tucson, Arizona using eight Sky Quality Meters (SQMs). In order to analyze the data in a consistent way for comparison, we created a standard procedure for reduction and analysis using python and MATLAB. The series of python scripts and MATLAB codes to remove faulty data and examine specifically anthropogenic light pollution and illustrate how the light pollution changes in relation to time, distance from the city, and airglow. Data are then analyzed by a recently developed sky brightness model created by Dan Duriscoe of the U.S. National Park Service. To quantify the measurements taken by SQMs, we tested the wavelength sensitivity of the devices used for the data collection. The findings from the laboratory testing have prompted innovations for the SQMs as well as given a sense of how data gathered by these devices should be treated. Dr. Shane Larson and I are implementing findings and procedures at Utah State University (USU), along with equipment acquired through the Undergraduate Research and Cre- ative Opportunities (URCO) grant to create a light-map of the university campus and surrounding city. Addi- tionally, the luminosity output of outdoor light fixtures will be analyzed via a Pocket Lux Light Meter. As USU has a sustainability program, Blue Goes Green, that has already implemented lighting codes, the goal of this project is the work with the collegiate administration to renovate old lighting fixtures

Anti-carcinogenic effects of exercise-conditioned human serum: evidence, relevance and opportunities

Regular physical activity reduces the risk of several site-specific cancers in humans and suppresses tumour growth in animal models. The mechanisms through which exercise reduces tumour growth remain incompletely understood, but an intriguing and accumulating body of evidence suggests that the incubation of cancer cells with post-exercise serum can have powerful effects on key hallmarks of cancer cell behaviour in vitro. This suggests that exercise can impact tumour biology through direct changes in circulating proteins, RNA molecules and metabolites. Here, we provide a comprehensive narrative overview of what is known about the effects of exercise-conditioned sera on in vitro cancer cell behaviour. In doing so, we consider the key limitations of the current body of literature, both from the perspective of exercise physiology and cancer biology, and we discuss the potential in vivo physiological relevance of these findings. We propose key opportunities for future research in an area that has the potential to identify key anti-oncogenic protein targets and optimise physical activity recommendations for cancer prevention, treatment and survivorship

Evaluating lethal toxicant doses for the largest individuals of an invasive vertebrate predator with indeterminate growth

The brown treesnake (Boiga irregularis) was accidentally introduced to Guam and caused severe ecological and economic damages. Acetaminophen is an effective, low-risk oral toxicant for invasive brown treesnakes, and an automated aerial delivery system (ADS) has been developed for landscape-scale toxic bait distribution. A fixed dose of 80 mg of acetaminophen within a tablet inserted into a dead neonatal mouse (DNM) was lethal for all brown treesnakes in previous trials; however, these trials did not include very large individuals which are difficult to acquire for testing. Because most reptiles continue to grow throughout their lifespan, a small number reach much greater than average body sizes. Here, we tested effectiveness of 80 mg acetaminophen DNM baits for unusually large brown treesnakes as they became available. Our results confirmed that an 80 mg dose is lethal for the vast majority of snakes on Guam, but efficacy starts to diminish around 200 g of body mass. We also tested an alternative mouse bait configuration with 160 mg of acetaminophen that could be incorporated into the ADS to improve control of unusually large snakes. The 160 mg dose is expected to be effective for nearly all female snakes; males grow much larger and additional methods will be needed for extraordinarily large individuals. We describe a full dose-response curve for brown treesnakes to acetaminophen tablets and estimate the LD90 at 299 mg/kg and the LD99 at 578 mg/kg. To our knowledge, this is the first published dose-response curve for an invasive vertebrate with indeterminate growth

NaBr Poisoning of Au/TiO\u3csub\u3e2\u3c/sub\u3e Catalysts: Effects on Kinetics, Poisoning Mechanism, and Estimation of the Number of Catalytic Active Sites

Sodium bromide was used to intentionally poison a commercial Au/TiO2 catalyst with the goals of understanding the nature of halide poisoning and evaluating the number and nature of the catalytic active sites. A series of eight poisoned catalysts were prepared by impregnating the parent catalyst with methanolic solutions of NaBr. Each catalyst was tested with CO oxidation catalysis under differential reactor conditions; O2 reaction orders and Arrhenius activation energies were determined for each material. All of the kinetic data, including a Michaelis−Menten analysis, indicated that the primary effect of adding NaBr was to reduce the number of catalytically active sites. Density functional theory calculations, employed to evaluate likely binding sites for NaBr, showed that NaBr binds more strongly to Au corner and edge atoms than it does to the titania support or to exposed Au face atoms. Infrared spectroscopy of adsorbed CO, along with a Temkin analysis of the data, was also used to evaluate changes to the catalyst upon NaBr deposition. These studies suggested that NaBr addition induces some subtle changes in the coverage dependent properties of CO adsorption, but that these did not substantially impact the CO coverage of the CO binding sites. The experimental and computational results are discussed in terms of possible poisoning mechanisms (siteblocking vs off-site binding and modification); the nature and number of active sites are also discussed in the context of the results