published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Medicine / Master / Master of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy

A digoxigenin-labeled nonradioactive detection system was used to screen a zebrafish cDNA library for p53-like and ras-like genes. One clone was isolated and identified as an incomplete p53-like gene. The insert size of this clone is 1777 bp, which encodes part of evolutionarily conserved region II and all of regions III, IV, and V. A magnetically enriched whole zebrafish cDNA library was constructed to enhance possible recovery of ras-like genes in zebrafish. One clone, termed Zras-Bl, carried an insert of 2592 bp with an open reading frame encoding a 188 amino acid residue ras p21 protein. Based on total protein sequence, this expressed zebrafish ras p21 is most closely related to human N-ras (91% homology), with lesser homology to Ha-ras (84%) and Ki-ras (85%). Preliminary partial sequence data obtained by genomic and reverase transcriptasepolymerase chain reaction (RT-PCR) screening indicate the presence of at least one additional expressed ras gene in zebrafish. The tumorigenicity and Ki-ras mutational properties of dietary 7,12-dimethylbenz[a]anthracene (DMBA) and dibenzo[a,l]pyrene (DBP) were compared in rainbow trout. Both chemicals elicited predominantly 12(1)G->A and 12(2)G->T mutations in trout liver tumors. Two {12(1)G->T and 12(2)G->T} and one {12(1)G->A and 12(2)G->T} double mutation were also observed in DBP livers tumors, but not in DMBA liver tumors. Some stomach tumors from both chemicals exhibited so much DNA degradation that routine PCR amplification was not possible. Among sixteen DMBA stomach tumors with intact DNA, no Ki-ras mutations were found. Of sixteen DBP stomach tumors examined, one had 12(1)G->A and two had 13(1)G->C mutations. The observed G->T transversions are compatible with apurinic mutagenesis driven by unstable DNA adducts arising from one-electron oxidation, but this is not true for the major G->A transitions or G->C transversions and rare double mutations found in this study. The low sensitivity of direct sequencing may limit the frequency of ras mutant detection in this study. / Graduation date: 1996

Aortic aneurysm is a complex disease manifesting in a localized dilation of the aorta developing over years and carries with it a significant chance of rupture resulting in death. As only surgical methods are currently available for treatment, there is a need to understand the underlying mechanisms of the disease and how they develop and lead to expansion and rupture. Thus, the study of the formation and progression of aneurysm has also focused on quantifying any changes observed in fiber realignment and altered mechanical properties leading to vascular disease. Animal models of aneurismal disease can be useful for studying alterations during disease development (e.g., in the tissue's mechanical response). Recent efforts have been aimed at determining both the biomechanical alterations that occur with aneurysm formation and their potential for rupture. However, previous animal model work is lacking quantitative descriptions of how biomechanical response and vessel remodeling change with time and lead to the diseased state. Thus, there is a need for determining an appropriate animal model for aneurysm and developing an adequate method for quantifying and determining disease progression through alterations in biomechanical response.

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Parkinson’s disease (PD) is the most common movement neurodegenerative disease affecting approximately 1% of the population over 60. Though originally thought to be sporadic in nature, a genetic component is increasingly being linked to the disease. Of these genes, mutations in DJ-1 (PARK7) cause early onset autosomal recessive PD. Initial workup of the DJ-1 protein has suggested that it may act in the cell by combatting oxidative stress though the mechanism by which it does so is unclear. Thus, though much work has attempted to elucidate a function at the biochemical, cellular and organismal level, the overt physiological role of DJ-1 remains elusive. In this dissertation, we explore the mechanisms through which DJ-1 confers neuroprotection, particularly in the case of oxidative stress insult. We demonstrate that DJ-1 acts through the pro-survival protein AKT to accomplish its neuroprotective function. Moreover, we note that DJ-1 likely serves its role as an antioxidant through the NRF2 master antioxidant regulator pathway a pathway that is, itself, likely to be regulated by AKT. Together, our results demonstrate that neuroprotection by DJ-1 is done through a signaling pathway involving both AKT and NRF2 and that disruption of the former in PD likely results in abolishing this signaling pathway. Finally, to generate a better animal model of PD, we demonstrate that backcrossing DJ-1 null mice - which originally did not demonstrate any gross histopathological or behavioral phenotypes – display unilateral dopaminergic degeneration that progresses to bilateral degeneration with aging, a feature reminiscent of classical PD progression. Collectively, this thesis takes a two-sided approach to address the biochemical and physiological functions of DJ-1 within the cell and the mouse in hopes of elucidating mechanisms of neuronal death to devise better translational therapies.

Ischaemic stroke is responsible for substantial death and disability and creates a huge financial burden for healthcare budgets worldwide. At present there are few effective treatments for acute stroke and these are urgently required. Increased understanding of the ischaemic cascade has generated interest in neuroprotection for focal cerebral ischaemia. However, treatment effects observed in of over 500 interventions in animal models have yet to be translated to the clinic. Systematic review and meta-analysis allows unbiased identification of all relevant data for a given intervention, gives a clearer view of its true efficacy and the limitations to its therapeutic potential. Understanding the reasons for this bench-to-bedside failure and providing quantitative explanations may help to address these discrepancies. Random effects weighted mean difference meta-analysis of six interventions (tirilazad, tPA, NXY-059, Hypothermia, Piracetam and IL1-RA) reported study quality to be consistently low. In some instances, potential sources of bias were associated with overestimations of efficacy. Likewise, clinical trials have tested interventions in conditions where efficacy was not observed in animals. Cumulative meta-analysis suggests that for tPA the estimate of efficacy is stable after the inclusion of data from 1500 animals; hypothermia and FK506 are the only other interventions to have been tested in at least 1500 animals. Meta-regression suggests biological rather methodical factors are better predictors of outcome; a major limitation of these data is the impact of publication bias, and this work suggests effect sizes from met-analyses are inflated by about 31% because 16% of studies remain unpublished. The systematic review and meta-analysis of hypothermia was used to plan experiments investigating the possible impact of pethidine, a drug used to prevent shivering. This in vivo experiment, in which potential sources of bias were minimised, suggests that pethidine does not influence the observed efficacy of hypothermia in an animal model of ischaemic stroke. This thesis reports that animal studies of ischaemic stroke are often not conducted with sufficient rigour. Both minimising potential sources of bias in individual experiments and using meta-analysis to summarise data from a number of experiments may be helpful in improving the translation of neuroprotective efficacy in ischaemic stroke.