Thesis (Ph. D.)--Open University. BLDSC no. DX175924.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3241. Adviser: Henry T. Wilkinson. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (M.S.)--Oklahoma State University, 2009. / Vita. Includes bibliographical references.

Thesis (M.S.)--Texas A & M University, 2003. / "Major Subject: Plant Pathology." Title from author supplied metadata (automated record created on Apr. 30, 2004.). Vita. Abstract. Includes bibliographical references.

Thesis (M.S.)--East Carolina University, 2009. / Advisor: John Heilmann. Title from PDF t.p. (viewed July 1, 2010). Presented to the faculty of the Department of Communication Sciences and Disorders. Includes bibliographical references.

Thesis (Ph. D.)--Texas A & M University, 2003. / "Major Subject: Plant Pathology." Title from author supplied metadata. Includes bibliographical references.

Thesis (M.S.)--Texas A & M University, 2003. / "Major Subject: Plant Pathology." Title from author supplied metadata. Includes bibliographical references.

Trypanosoma brucei is a protozoan parasite, and the causative agent of Human African Trypanosomiasis and Nagana in cattle. The life cycle and cell cycle of the parasite is complex and unusual. In particular, cytokinesis regulation in T. brucei is divergent, and significantly does not involve the formation of an actomyosin contractile ring; instead, a furrow ingresses longitudinally along the cell following the axis of the subpellicular microtubules which form a cytoskeletal cage around the cell body. As the organelles are positioned longitudinally in the posterior half of the cell, and in different positions according to life cycle stage, the cleavage axis is therefore subject to a number of constraints which must be overcome for symmetrical allocation of organelles to the daughter cells. It is highly likely that the subpellicular microtubule cytoskeleton plays important roles in cytokinesis furrow ingression. Presumably this process must involve microtubule and membrane remodelling at the site of the furrow apex to form the two discrete daughter cell bodies, and this is likely to require changes in microtubule dynamics, at least locally. Additionally, the cytoskeleton could influence the position of the cleavage plane by denoting polarity, or the timing of furrow initiation through mechanosensing. The divergent nature of T. brucei cytokinesis implies that regulators of this process could be exploited as a source of potential novel drug targets. The Polo-like kinase, PLK, has previously been shown to be required for furrow ingression during cytokinesis in bloodstream form T. brucei. This study aimed to further our understanding of the regulation of cytokinesis by PLK by investigating how its activity is regulated in vitro and in vivo. In other organisms, PLK is known to influence microtubule dynamics, and given the likelihood that microtubule dynamics are important for furrow ingression in T. brucei, this study also aimed to investigate the role of the cytoskeleton in cytokinesis. An orthologue of a microtubule-associated protein required for cytokinesis in plants, AIR9, was functionally characterised in T. brucei, and the role of subpellicular microtubules in cytokinesis was investigated via the use of microtubule inhibitors. Soluble and active recombinant PLK was purified from E. coli as a 6X Histidine fusion protein (6XHis:PLK). 6XHis:PLK autophosphorylated prolifically, and removal of these phosphorylations with lambda protein phosphatase significantly reduced the ability of 6XHis:PLK to transphosphorylate generic kinase substrates. Further, the importance of a conserved threonine residue (T198) in the T-loop of T. brucei PLK, which is a major site for regulation by upstream kinases in other organisms, was investigated. Substitution of T198 with a non-polar (alanine or valine) or a phosphomimetic (aspartic acid) residue revealed that this residue was important for PLK activity in vitro. However, expression of T198 variants in vivo showed T198V and T198D to be functional, suggesting that PLK activity is not regulated in vivo by phosphorylation at this site. The role of the polo box domain (PBD) of PLK in regulating PLK activity was also investigated. In PLKs from other organisms, the PBD autoinhibits PLK activity. Here, while recombinant 6XHis:PBD could pull down full length ty:PLK from T. brucei lysates, kinase assays indicated that the PBD did not inhibit the activity of full length PLK. Thus, regulation of the activity of PLK in T. brucei appears to be divergent. Functional characterisation of T. brucei AIR9 by RNAi mediated depletion revealed roles for this protein in the relative positions of organelles and the position of the cleavage plane in both life cycle stages. However, the phenotypes observed during RNAi experiments differed between procyclic and bloodstream form parasites. For procyclic form parasites, the defective cytokinesis resulting in non-equivalent progeny seemed to occur in cells with organelle positioning defects suggesting that problems with cytokinesis were secondary to the major organelle positioning defect. Abnormal organelle positioning was far less frequent in bloodstream form parasites, which none the less seemed impaired in the accurate positioning of the cleavage axis. AIR9 was shown to localise to the cytoskeleton of bloodstream and procyclic trypanosomes using two different epitope tagging approaches, and following induction of AIR9 RNAi, AIR9 was preferentially depleted from the posterior end of the cell, supporting a designation of AIR9 as a microtubule-associated protein. However, data do not support a role for AIR9 in cytoskeletal stability, since transmission electron microscopy showed the structure of cytoskeletal microtubules was not affected following depletion of AIR9. Hence, I suggest that AIR9 is more likely to be a scaffold protein potentially involved in the integration of signalling pathways to control cell polarity and cytokinesis. This study also demonstrated through the application of inhibitors of microtubule dynamics that microtubule dynamics are important for cytokinesis in T. brucei. Vinca alkaloid treatment of bloodstream form parasites arrested furrow ingression, while taxol inhibited initiation of cytokinesis in bloodstream form parasites and affected cleavage plane positioning in procyclic cells. In addition, organelle positioning was inhibited through the application of the vinca alkaloid, vinblastine, to procyclic form cells. Thus, although these studies indicate that there are differences in the cytoskeleton makeup of bloodstream and procyclic trypanosomes, the data obtained are consistent with microtubule dynamics playing crucial roles in cell polarity and cytokinesis in both life cycle stages.

Overall paediatric high grade glioma (pHGG) has a poor prognosis, in part due to the lack of understanding of the underlying biology. We therefore used high resolution 244K oligo array comparative genomic hybridisation (oligo aCGH) (Agilent Technologies) to analyse DNA from 38 formalin-fixed paraffin embedded pHGG samples, including 13 DIPG. The pattern of gains and losses were distinct from those seen in HGG arising in adults. In particular we found 1q gain in 21% of our cohort compared to 9% in adults. Homozygous loss at 8p12 was seen in 6/38 (15%) of pHGG. This deletion has not been previously reported in adult or paediatric high grade gliomas. The minimal deleted region is of the gene ADAM3A and homozygous deletion of ADAM3A was confirmed by quantitative real time PCR (qPCR). This novel homozygous deletion of ADAM3A in pHGG merits further study. Amplification of the 4q11-13 region was detected in 8% of cases and included PDGFRA and KIT and subsequent qPCR analysis was consistent with amplification of PDGFRA. MYCN amplification was seen in 2/38 samples (5%) and was shown to be significantly associated with anaplastic astrocytomas (p=0.03). Loss of CDKN2A/B was seen in 4/38 (10%) samples by oligo a CGH, confirmed by FISH on TMAs, and was restricted to supratentorial tumours. ~50% of supratentorial tumours were positive for CDKN2B expression by IHC, whilst ~75% of brainstem gliomas were positive for CDKN2B expression (p = 0.03). Overall DIPG shared a similar spectrum of changes to supratentorial HGG with some notable differences including high frequency of 17p loss and 14q loss, low occurrence of 10q loss and lack of CDKN2A/B deletion.

Whilst the acute effects of traumatic brain injury (TBI) may be devastating, growing evidence suggests TBI may initiate long-term neurodegenerative processes. Indeed, an epidemiological association between TBI and Alzheimer’s disease (AD) has been demonstrated. Here the link between TBI and chronic neurodegeneration is explored by examining associated pathologies in material from patients surviving TBI for varying intervals. Furthermore, the Aβ clearing enzyme, neprilysin, has emerged as important with regards to Aβ dynamics in AD and thus may be relevant to Aβ-plaque formation in TBI. Its influence on Aβ dynamics in TBI was explored in an animal model of TBI and in human TBI material. First, a potential association between acute-Aβ plaques post-TBI and a GT repeat polymorphism of the Aβ-degrading enzyme, neprilysin was investigated using DNA fragment sizing analysis (n=81). Results show there was an increased risk of Aβ plaques for patients with greater than 41 total repeats (p<0.0001, OR:10.1). In addition, the presence of 22 repeats in at least one allele was independently associated with plaque deposition (p=0.03, OR: 5.2). In contrast, the presence of 20 GT repeats in one allele was independently associated with a reduced incidence of Aβ plaques (p=0.003). These data suggest a genetically linked mechanism that determines which TBI patients will rapidly form Aβ plaques. The role of neprilysin in TBI was further examined using immunohistochemistry (IHC) to evaluate plaque formation in neprilysin knock-out mice at 2h, 48h and 4mo following a single controlled cortical impact in mice. Young (2mo) and older (8mo) mice were examined at each time point and compared to age-matched injured and uninjured wild-type mice (n=4 for all groups). No plaques were observed at any time point or in age group indicating that neprilysin deficiency alone is insufficient to recapitulate the plaque pathology observed in humans using this model. Next, post-mortem brains from long-term survivors of just a single TBI (1 to 47 years survival; n=39) versus uninjured, age-matched controls (n=47) were examined for hallmark AD pathologies, neurofibrillary tangles (NFTs) and Aβ plaques using IHC and thioflavin-S staining. NFTs were exceptionally rare in young, uninjured controls, yet were abundant and widely distributed in approximately one third of TBI cases. Moreover, thioflavin-S staining revealed that while plaque-positive control cases displayed predominantly diffuse plaques, 64% of plaque-positive TBI cases, displayed predominantly thioflavin-S positive plaques or a mixed positive / diffuse pattern. These data indicate that TBI may induce or accelerate the presence of neurodegeneration-associated pathologies many years following just a single injury. To explore potential mechanisms driving plaque formation post-TBI, IHC was used to examine amyloid-β’s precursor, APP in TBI cases with acute survival (10h to 1 week; n=20), moderate survival (1 week to 1 year; n=17) and long-term survival (>1 year; n=33), versus uninjured controls (n=47). Following TBI, increased somatic APP immunoreactivity (IR) was observed in all TBI cases (n=70; 100%) versus (n=47; 70%) of controls (Chi Sq; p=0.00001). Moreover, increased APP IR was present in all survival intervals examined, (10h to 10 days, p=0.001; 10 days to 1 year, p=0.0001; > 1 year; p=0.001; all Chi Sq) versus controls, and an increased extent / distribution of pathology was demonstrated in the TBI group regardless of survival time. Interestingly, APP IR was not associated with the presence of plaques suggesting increased APP alone is insufficient to drive post-traumatic plaque formation. The Tar-DNA binding protein, TDP-43 has emerged as an important pathological feature in various chronic neurodegenerative diseases including frontotemporal lobe dementia, and more recently in dementia pugilistica caused by repetitive TBI. Here we examined survivors of a single TBI using IHC specific for TDP-43. TBI cases were acute (n=23: Survival <2 weeks) and long-term (n=39; 1-47 years survival) and were compared to uninjured controls (n=47). No association was found between single TBI and pathological TDP-43 inclusions. Specifically, just 3 of 62 TBI cases displayed p-TDP-43 pathology versus 2 of 47 control cases. However, IR to phosphorylation-independent TDP-43 was commonly increased in the cytoplasm following TBI with both acute and long-term survival, potentially as part of a normal stress-response. Finally, acute and chronic inflammation was evaluated in the corpus callosum following TBI, a region highly susceptibility to injury. IHC specific for CR3/43 and CD68 was analysed using digital quantitation to determine the percentage area of positive staining in acute TBI cases (survival < 14 days; n=18) and compared to age-matched controls (n=18). Long-term survivors of TBI (surv >1 year; n=23) were compared to controls (n=23) and a further subset of acute TBI cases (survival < 10 days; n=12) compared to age-matched long-term survivors of TBI (survival >1 year; n=12). While no quantitative difference was demonstrated between groups, 44% of long-term survival TBI cases displayed extensive amoeboid cells versus 0% of controls (Chi Sq; p=0.0004), indicating a percentage of TBI patients may develop a chronic inflammatory response in a time-frame consistent with epidemiological associations linking TBI with AD.