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.

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.

Today genetic services including genetic counselling are widespread across the world. Although developing countries, like Colombia, have started to apply genetic knowledge to the health area, genetic counselling is usually integrated in the routine clinical genetic consultation, however, before this study the process of communication involved in it had not been explored. In collaboration with the Colombian Association of Medical Genetics, the Bogotá Health Service, and the University of Warwick (UK), I observed 25 genetic consultations in five Colombian genetic clinics. I undertook semi-structured interviews with patients / families before and after the consultation. Thematic analysis of the interview transcripts established mismatches between physician perception and patient comprehension. Efficient communication was affected by patient, relatives, practitioner and external factors. Among these environmental factors were excessive administrative procedures, interruptions during the encounter, patients‟ lack of interest to medical terminology, doctors using scientific language, excessive information given in one session, beliefs and education level of the patient and/or relatives, patient distress caused by bad news, unfulfilled expectations and no availability/accessibility of treatment. I also interviewed 20 medical practitioners working in genetics services. There was general agreement that genetic counselling in Colombia was challenging, and that more training in communication skills was required at Medical schools at undergraduate and postgraduate level. Many physicians did not believe that other health professionals should work as genetic counsellors. There was a general recognition of limited genetic knowledge, awareness and understanding in most medical specialities. These results have made a valuable contribution to describe the current situation with genetics consultation and counselling in Colombian genetic clinics, and have already influenced the future development of an effective and robust genetic counselling service in Colombia. They will also be used in the development of the academic curriculum related to basic and clinical genetics at Colombian Universities.

The bHLH protein TAL1 (SCL) is a critical regulator of vertebrate hematopoiesis and is misregulated in T-cell acute lymphoblastic leukemia (T-ALL). This thesis studied chromatin looping interactions at the TAL1 locus – defining the first structural model which accounts for a number of phenomena associated with TAL1, its flanking genes and its relationship with its functional paralogue LYL1. The chromosome conformation capture (3C) and its high-throughput variant 4C-array technologies have been applied to characterise the chromatin interactions. Intriguing chromatin organisations have been identified at the TAL1 and LYL1 loci, which are closely associated with transcriptional regulation, chromosomal abnormality and regulatory remodelling through evolution. Firstly, in TAL1 expressing cells, the locus adopts a “cruciform” configuration – forming an active chromatin hub which brings together the TAL1 promoters, its stem cell and erythroid enhancers, and two CTCF/Rad21-bound insulators. Secondly, loss of a GATA1-containing complex bound by the TAL1 erythroid enhancer and its promoter is sufficient to disrupt the formation of the hub and the entire cruciform structure and results in decreased TAL1 expression. Thirdly, it demonstrates that genes flanking TAL1 are also dependent on this hub and that TAL1 promoters interact directly with intron 1 of the neighbouring STIL gene. This TAL1/STIL interaction also provides a structural link between the DNA sequences which mediate micro-deletions in 25% of cases of T-ALL. Finally, it demonstrates that a GATA1-dependent chromatin looping mechanism also exists at the LYL1 locus which is strikingly similar to that mediating contact between the TAL1 promoter and its erythroid enhancer. Conservation of core chromatin looping at the TAL1 and LYL1 loci may account for some aspects of their functional relationships. It also suggests that looping mechanisms at both loci could also facilitate cis-regulatory maintenance and/or remodelling during vertebrate evolution.

Gene therapy has a great potential for the treatment of a wide range of diseases. However, the development of a safe and efficient delivery vector is the major obstacle for gene therapy. Recently synthesized 2 - (dimethylamino) ethyl methacrylate 2-(methacryloxloxyethyl phosphorylcholine) (DMA-MPC) diblock copolymer was investigated in this work as a novel non-viral vector for gene delivery. It has been previously demonstrated that the cationic DMA block can condense DNA efficiently. The zwitterionic PC head groups are found naturally in the outer leaflet of biomembranes and are extremely biocompatible. It is thus proposed here that the MPC can act as a new steric stabilizer to the system. Different compositions of DMA-MPC diblock copolymers were evaluated. The MPC block with minimum length 30 monomeric units can successfully provide steric stabilization to the system, and reduce nonspecific cellular interaction by providing a steric barrier to the DNA complexes. However, long MPC chain can hinder the interaction between cationic DMA and DNA, leading to the formation of loosely condensed complexes which were more susceptible to enzymatic degradation. Therefore the composition of the copolymer must be carefully adjusted so that the DNA condensing and steric stabilization effect are well balanced. In order to investigate the cellular uptake mechanism DMA homopolymerDNA complexes, the effect of different endocytosis inhibitors was examined. Microtubules and actin filaments were involved in the uptake of DNA complexes, suggesting that the complexes were internalised by endocytosis. Both the clathrin- and caveolae- mediated pathway were responsible for the uptake of DNA complexes, and the former appeared to be the main route of entry. Finally, folic acid ligand was incorporated into the DMA-MPC copolymer in order to improve the specific targeting. Initial data showed that there was selective uptake of the folate conjugated system in folate receptor expressing cells possibly via receptor mediated endocytosis. However, parameters such as the optimum length of MPC component, number of ligands per DNA complex and the composition of the system need to be further investigated in order to maximize the specificity and transfection efficiency.

The aims of this study were to (1) evaluate cellular senescence in chondrocytes from osteoarthritic articular cartilage, (2) investigate the hypothesis that oxidative stress is a feature of canine OA chondrocytes and that oxidative stress contributes to cellular senescence in canine chondrocytes, (3) investigate the hypothesis that osteoarthritic chondrocytes alter the gene expression of adjacent normal chondrocytes in OA joints leading to modulation of genes known to play a role in the pathogenesis of OA and (4) evaluate the presentation of dogs undergoing femoral head excision in veterinary referral practice in the UK as a treatment for osteoarthritis of the coxofemoral joint, and to categorise the distribution and severity of associated pathological lesions. Chondrocytes from osteoarthritic and normal cartilage were examined for levels of senescence. Initially chondrocytes were cultured using an alginate bead culture system, thought to mimic the extracellular matrix of articular cartilage. However, these chondrocytes showed almost no growth as compared to monolayer culture where they grew rapidly. OA chondrocytes entered the senescent state after 1.5 to 4.9 population doublings in monolayer culture, while normal chondrocytes underwent 4.8 to 14.6 population doublings before entering the senescent state. Osteoarthritic chondrocytes had increased levels of markers of cellular senescence (senescence associated beta-galactosidase accumulation and p16 protein accumulation) as compared to normal chondrocytes, suggesting that chondrocyte senescence is a feature of canine osteoarthritis. An experimental model for the induction of oxidative stress in chondrocyte cell culture was developed using tert-Butyl hydroperoxide and total cellular glutathione was measured as an indicator of cellular oxidative stress levels. Experimental induction of oxidative stress in both normal and osteoarthritic chondrocytes in cell culture resulted in increased amounts of cellular senescence, shown by an increase in levels of senescence associated beta-galactosidase accumulation and decreased replicative capacity. Experimental induction of oxidative stress also resulted in altered gene expression of three genes important to the degradation of the extracellular matrix; MMP-13, MMP-3 and Col-3A1, measured by RT-PCR, in normal canine chondrocytes in monolayer cell culture. MMP-3 showed the greatest relative expression change, with a fold-change of between 1.43 and 4.78. MMP-13 had a fold change of 1.16 to 1.38. Col-3A1 was down regulated, with a fold-change of between 0.21 and 0.31. These data demonstrate that experimentally induced oxidative stress in chondrocytes in monolayer culture increases levels of cellular senescence and alters the expression of genes relevant to the pathogenesis of canine OA. Coculture of osteoarthritic chondrocytes with normal canine chondrocytes resulted in gene modulation in the normal chondrocytes. Altered gene expression of ten genes known to play a role in the pathogenesis of osteoarthritis was detected in the normal chondrocytes (fold change shown in brackets); TNF-alpha (11.95), MMP-13 (5.93), MMP-3 (5.48), IL-4 (7.03), IL-6 (5.3), IL-8 (4.92), IL-F3 (4.22), COL-3A1 (4.12), ADAMTS-4 (3.78) and ADAMTS-5 (4.27). In total, 594 genes were significantly modulated suggesting that osteoarthritic chondrocytes contribute to the disease propagation by altering the gene expression of adjacent normal chondrocytes, thus recruiting them into the disease process. Gene expression changes were measured by microarray analysis and validated by RT-PCR and Western blot analysis. An epidemiological study of femoral heads collected from dogs undergoing total hip replacement surgery as a treatment for osteoarthritis of the coxofemoral joint secondary to canine hip dysplasia revealed that there was no characteristic pattern of cartilage lesion for canine hip dysplasia. Severe pathology of the femoral head with cartilage erosion occurred in 63.9% of cases and exposure of subchondral bone in 31.3% of cases. The work presented in this thesis has demonstrated that cellular senescence is a feature of chondrocytes from canine osteoarthritic cartilage and suggests that cellular senescence and oxidative stress play an important role in the pathogenesis of osteoarthritis in dogs.

Improving the differentiation capacity of stem cells by defining the ideal physico-chemical and spatial parameters will enhance the efficiency of stem cell-based clinical therapies. In this thesis the hypothesis that heat shock (elevated temperatures) could positively influence osteogenic differentiation was investigated. A methodology was developed to employ magnetic nanoparticle-based local heating (magnetic hyperthermia) to spatially control temperature distribution at the cellular level. For this purpose, bacterially synthesized zinc and cobalt doped iron oxide nanoparticles with tuned physical and magnetic properties were assessed for their interaction with cells, and their magnetic response and heating properties when in a cellular milieu. Nanoparticles with moderate levels of zinc doping showed strong heating effects and minimal cytotoxicity, proving to be promising candidates for cellular applications. The effects of mild and severe heat stress were assessed by heating cells using conventional techniques such as water baths (bulk heat shock); using localised heating with extracellular nanoparticle suspensions; or by targeting different cellular regions with nanoparticles. The effect of the heat shock treatment on the osteogenic differentiation was assessed in primary bone marrow-derived human mesenchymal stem cells and a cancerous osteoblastic cell line, MG-63. With both bulk and nanoparticle-mediated extracellular mild heat shock (~42°C), very little evidence for a positive effect on osteogenic differentiation was found in both cell types. On the other hand, severe heat shock treatments (>50°C) showed differentiation enhancement though this also negatively impacted cell viability. Further experimentation can shed light on the optimum temperature range within which the differentiation behaviour of cells can be influenced without compromising viability. The nanoparticle-based methodology developed here to apply heat shock to different cellular components, could lead to further work investigating how intracellular pathways translate the heat stimulus into a cellular response for stem-cell based applications.

No description available.

Women are up to twice as likely to suffer from post-traumatic stress disorder (PTSD) than men. Failure to discriminate between cues predicting threat and safety is associated with PTSD, yet sex differences in fear discrimination remain poorly understood. Here, we examined sex differences in auditory fear discrimination in rats using a combination of behavioural, computational and electrophysiological methods. In the initial behavioural study, males and naturally cycling females underwent 1-3 days of discrimination training, consisting of pairings of one tone (CS+) with shock and presentations of another tone (CS-) alone. After one day of training, females, but not males, discriminated between the CS+ and CS-. With 2-3 days of training, however, males discriminated and females generalised between the CS+ and CS-. Further testing also revealed that males successfully encode the CS- as a safety signal, whereas females do not. Using reduced computational models, we investigated how both ‘discrimination’ and ‘generalisation’ phenotypes can be generated in silico. We achieved this through a simulation of neural activity produced via ‘fear’ and ‘safety’ neural sub-populations of the basolateral amygdala (BLA) in response to CS+ and CS- cues. By using a model representation of extended fear discrimination training and retrieval, we found that generalisation between the CS+ and CS- could be produced from reduced inhibition, or increased excitation, of fear neurons. Due to their involvement in regulating learned fear, we additionally aimed to investigate the roles of the prelimbic (PL) and infralimbic (IL) cortices of themedial prefrontal cortex in fear discrimination. By concurrently recording activity from the PL, IL and BLA in awake behaving animals during retrieval of the CS+ and CS- after extended discrimination training, we examined the individual contributions and functional interactions of these regions during this learning paradigm. We found that, in males, the PL showed an increase in power at both theta (4-12 Hz) and gamma (30-120 Hz) frequencies during presentations of the CS- compared to the CS+, whereas this increase was largely absent in females. Taken together, these results indicate that, while females show fear discrimination with limited training, they generalise with extended training. We hypothesised that this generalisation in females is likely due to impaired safety learning, which may result, in part, from sex differences in the neural circuitry underlying fear discrimination.

Colorectal cancer (CRC) is the second most common cancer in Europe, with the second highest mortality rate. Although prognosis is improving, survival rates remain poor for those presenting with the most advanced stages of the disease. There is therefore a need for improved early diagnosis and thus a greater understanding of the early stages of the development of colorectal tumours is desirable. Additionally, as most deaths in colorectal cancer are due to advanced metastatic disease, it is of great interest to explore any potential mechanisms by which metastatic disease can be inhibited. N-WASP is a ubiquitously expressed protein with multiple intracellular roles including actin regulation and maintaining stability of epithelial cell-cell junctions. Through its role as an actin regulator, it has been implicated in the processes of invasion and metastasis of multiple cancer types. Its role in the development and progression of colorectal cancer however has not been fully explored. This thesis will present a series of in vitro and in vivo studies that were carried out with the aim of answering the following questions: • Does N-Wasp have a role in normal intestinal homeostasis? • Does N-Wasp knockout affect the development of tumours in a mouse model of intestinal tumourigenesis? • Does N-Wasp knockout affect the invasive properties of intestinal cancer in vitro? • Does N-WASP correlate with prognosis or other indicators in human colorectal cancer TMAs? Findings from the in vivo experiments, using an inducible, gut-specific knockout model, have uncovered potential roles for N-Wasp in regulating differentiation and migration of intestinal epithelial cells. Although it had no effect in short term models of intestinal hyperproliferation, N-Wasp knockout increased tumour burden and decreased survival in an established in vivo model of intestinal tumourigenesis, in which there is heterozygous loss of Apc (Apcfl/+). No effect was seen on tumour development or survival when additional N-WASP knockout was introduced into a more rapid model, with heterozygous loss of Apc and mutation of Kras (Apcfl/+ KrasG12D/+). N-WASP expression in human colorectal cancer was assessed using immunohistochemical staining of two tissue microarrays. Low levels of N-WASP expression were found to be associated with presence of MMR deficiency. There was no statistically significant difference in overall or cancer specific survival based on N-WASP expression. Collectively, the data presented here suggest a previously unreported role for N-WASP in regulation of intestinal epithelial differentiation and indicate that it may act as a tumour suppressor against development of benign precursor lesions of colorectal cancer. Further research is warranted to delineate the mechanisms underlying these processes.