Pathology - Theses
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ItemTherapeutic effects of copper bis(thiosemicarbazone) complexes in Alzheimer’s and Parkinson’s diseases( 2010)Neurodegeneration is a complex process and one that involves a myriad of physiological changes leading to chronic pathological states. Examples of neurodegenerative disorders include Alzheimer’s disease (AD) and Parkinson’s disease (PD). In this thesis, a group of compounds, CuIIbis(thiosemicarbazones) or CuII(btsc), was investigated for their ability as therapeutic agents in AD and PD. The CuII(btsc) compounds include CuII(atsm) and CuII(gtsm), and have been identified to be good candidates for CNS drugs due to their ability to be bioavailable and cross the blood brain barrier. In addition, they also possess unique properties that help target pathologies in both AD and PD. CuII(gtsm) administration to AD transgenic mice increased intracellular copper bio-availability and inhibited glycogen synthase kinase-3β (GSK-3β) through activation of an Akt signalling pathway. CuII(gtsm) also decreased the abundance of Aβ trimers and phosphorylated tau, and restored performance of AD mice in the Y-maze test to levels expected for cognitively normal animals. Improvements in Y-maze cognition correlated directly with decreased Aβ trimer levels. Therefore, therapeutic ability of CuII(gtsm) in the transgenic mice demonstrated that increasing intracellular copper bio-availability can restore cognitive function by inhibiting the accumulation of neurotoxic Aβ trimers and phosphorylated tau. CuII(atsm), on the other hand, was shown to be therapeutic in PD models by inhibiting nitrosative stress, in particular peroxynitrite (ONOO-) driven α-synuclein aggregation. Treatment of CuII(atsm) in four different PD animal models resulted in significant reductions in α-synuclein nitration and oligomerisation within the substantia nigra. In all models a significant increase in the survival of dopaminergic neurons was observed, indicating treatment was neuroprotective. Motor function was also improved in all models; this was consistent with improved dopamine metabolism as indicated by increased levels of tyrosine hydroxylase within the substantia nigra and vesicular monoamine transporter2 in the striatum. The findings from this thesis establish the therapeutic effects of CuII(btsc) compounds in animal models and suggest that these compounds could be effective disease modifying therapeutic agents for neurodegeneration in clinical studies.
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ItemCharacterization and therapeutic treatment in a transgenic mouse model of Amyotrophic Lateral Sclerosis( 2010)Amyotrophic Lateral Sclerosis (ALS) is a devastating adult–onset fatal neurodegenerative disorder that is characterized by loss of cortical and spinal motor neurons. This results in the clinical characteristics of muscle wasting and spasticity leading to paralysis and eventually death through respiratory failure within a period of 2-5 years from symptom onset. The aetiology of ALS is poorly understood and the neurodegenerative processes involved in ALS progression are diverse and complicated. Mutations in the superoxide dismutase (SOD1) gene are associated with a subset of familial ALS cases. There is considerable evidence implicating oxidative stress as a central mechanism in motor neuron death. However, the exact mechanism by which mutant SOD1 leads to motor neuron death has not been defined. Recent studies have shown that mutations in TDP-43 are linked to motor neuron degeneration in ALS, thus implicating TDP-43 as a new key player in ALS pathogenesis. How TDP-43 and SOD1 interact in ALS is not understood. ALS animal models have been developed to investigate the ALS disease processes. Overexpression of mutant SOD1 in mice has been found to imitate most of the disease phenotype observed in ALS patients. However this model has an aggressive phenotype which may complicate comparisons with slower progressing human ALS. The studies described in this thesis were designed to characterize a low copy transgenic SOD1G93A (TgSOD1G93A) mouse model by examining its behavioral aspects and protein markers associated with ALS. These mice may potentially provide a more appropriate model of human ALS. After characterization, we examined the efficacy of a novel potential therapeutic metallo-complex (bis(thiosemicarbazone-copper complex) (btsc) against disease progression in the TgSOD1G93A low copy mice. Treated mice were analyzed for behavioral, biochemical and immunohistochemical changes. TgSOD1G93A low copy mice were first characterized at different stages of their disease progression. Onset of TgSOD1G93A low copy mice disease symptoms was identified by significant body weight loss at 182 ± 2.3 days, followed by motor impairment at 205 ± 2.0 days and end stage disease at 263 ± 2.9 days. Significant motor neuron loss was apparent at 160 days through histochemical analysis of mouse lumbar spinal cord. Protein carbonyl (oxidative stress marker) levels were observed to be significantly elevated from symptom onset at 200 days. In addition, astrocytes and microglia activation were apparent throughout the 200 and 270 (end stage) day old spinal cord. After characterization of the low copy TgSOD1G93A model, animals were treated with potentially therapeutic CuII (btsc) metallo-complexes, (CuII(atsm) and CuII(gtsm)). CuII(atsm) treatment significantly extended survival, delayed disease onset and slowed disease progression in the TgSOD1G93A low copy mice. These therapeutic effects were associated with an increase in motor neuron survival at 200 and 230 days and a significant reduction in oxidative stress levels observed in both 230 day and end stage mouse spinal cord. In addition, astrogliosis was significantly attenuated. CuII(gtsm) treatment showed marginal improvement in survival in a small group of TgSOD1G93A low copy mice. Pathological changes to TDP-43 was examined at different disease progression stages in the TgSOD1G93A low copy and compared to end stage TgSOD1G93A high copy mice. In cytoplasmic fractions of spinal cord, high levels of phosphorylated full length TDP-43 and C-terminal fragment levels were present from pre-symptomatic (130 days) to end stage day old TgSOD1G93A low copy mice but absent in the end stage TgSOD1G93A high copy mice. Further evidence of altered TDP-43 homeostasis was reflected in positive cytoplasmic TDP-43 staining observed in symptomatic (200 day) and end stage TgSOD1G93A low copy mice. These results are consistent with published literature which reported that TDP-43 undergoes cleavage and phosphorylation, resulting in motor neuron death. Therefore, we examined the effects of CuII(atsm) treatment in the TgSOD1G93A low copy mice and observed decreased phosphorylated TDP-43. In conclusion, this thesis has characterized the TgSOD1G93A low copy mouse and demonstrated that a potential therapeutic compound for treatment of ALS, CuII(atsm), has robust protective effects. CuII(atsm) substantially increased animal survival and inhibited oxidative stress and TDP-43 pathological processing in the TgSOD1G93A low copy mice. These studies have important implications for the development of novel therapeutic approaches to treat ALS.
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ItemGenetic determinants of mammographic density as a risk factor for breast cancer( 2010)Background: Mammographic density (MD) for age and BMI is a strong risk factor (up to a 6 fold increase across extreme quantiles) for breast cancer. More than 60% of MD variation is estimated to be due to heritable (genetic) factors. Taking two different approaches, this work aimed to determine some of these genetic factors. The recently identified common genetic variants associated with small gradients in breast cancer risk and candidate common genetic variants identified using a genome wide association study (GWAS) of MD extremes were tested for an association with MD. Methods: Germline DNA extracted from peripheral blood samples from 497 monozygotic (MZ) and 330 dizygotic (DZ) twin pairs, and 634 of their sisters from 903 families were genotyped for 22 independent variants (12 from associations with breast cancer and 10 from GWAS of MD extremes). Mammographic dense area, percent dense area and non-dense area were measured by three observers using a computer thresholding technique. Associations with MD measures adjusted for age, BMI and other determinants were estimated: (a) cross-sectionally using a multivariate normal model for pedigree analysis (P-values reported by Px), and (b) between-sibships and (c) within-sibships using orthogonal transformations of outcomes and exposures. A combined test of association (P-values reported by Pc) was derived using the independent estimates from (b) and (c). The distributions of P-values across variants were tested for a deviation from the uniform distribution (P-values reported by Pu). Results: For the breast cancer associated common genetic variants tested, for dense area and percent dense area, the distributions of Pc-values deviated from the uniform distribution (both Pu<0.007), providing strong evidence that at least one genetic variant is associated with these MD measures. Consistent with their breast cancer associations, rs3817198 (LSP1) and rs13281615 (8q) were associated with dense area and percent dense area (all Px and Pc<0.05), and rs889312 (MAP3K1), rs2107425 (H19) and rs17468277 (CASP8) were marginally associated with dense area (some Px or Pc <0.05). For the candidate genetic variants from the GWAS of MD extremes the distributions of Pc-values deviated from the uniform distribution for dense area and percent dense area (Pu=0.07 and 0.009). One variant, rs10827227 (NRP1) showed strong evidence for an association with both dense area and percent dense area (Pc<0.009). For both approach, all associations were independent of menopausal status. Conclusion: At least two common breast cancer susceptibility variants and one common variant identified through a GWAS for MD extremes were associated with MD measures that predict breast cancer. Together these variants explain about 1% of the variation in MD.