Multi-component reaction (MCR) provides an easy and rapid access to libraries of organic compounds with diverse substitution patterns. This project aims to employ MCR for synthesis of both combinatorial (CL) and dynamic combinatorial (DCL) libraries for lead discovery.

Prion diseases are curious neurodegenerative diseases characterized by the conversion of a cellular protein, PrPC, into an infectious isoform, PrPSc. One of the earliest hallmarks of disease and concurrent with prion deposition, is the activation of the brain’s principal immune effector cells, microglia. In prion disease, activated microglia synthesize fairly low levels of pro-inflammatory cytokines, presumably to ameliorate the severe pathology that can arise in host tissue as a result of an acute inflammatory response. The specific stimuli and signaling pathways that lead to this modulation of function are as yet unknown. However, the involvement of miRNAs, a recently identified class of regulatory molecules, is likely. Recently, miR-146a was found to be upregulated in the brains of prion infected mice. In addition, its expression was found to be enriched in cells of microglial origin. It was hypothesized that, given the immunomodulatory role ascribed to miR-146a in macrophages, upregulation of miR-146a may function to attenuate the microglial immune response to prion infection. The first objective was to identify inflammatory related miRNAs associated with prion disease in microglia. Using Taqman Low Density Arrays, allowing for the detection of hundreds of miRNAs at once, the miRNAs of microglia treated with inflammatory agonists were profiled. The miRNA profile of activated microglia was found to be similar to that of macrophages. Furthermore, among the miRNAs profiled, miR-146a and miR-155 were the most highly induced and persistently expressed over 24 hours. The second objective was to investigate miR-146a induction. Therefore, microglia were treated with various agonists and miR-146a expression was determined using Taqman miR-146a assays. Although treatment with a PrP-mimic did not induce miR-146a expression, stimulation of TLRs 1, 2, 4, and 5, resulted in significant over-expression similar to what has been described previously. Moreover, in contrast to the rapid and transient induction of inflammatory mediators, miR-146a follows alternate kinetics functioning to prolong the dampening of the innate immune response following activation via TLR4 and TLR2. By employing a functional proteomic strategy, the third objective was to identify miR-146a regulated proteins. First, miR-146a expression was manipulated using miR-146a mimics and miR-146a inhibitors. Secondly, the functional model was validated by confirming decreased expression of IL6 by ELISA in miR-146a over-expressing microglia cells. Lastly, using Tandem Mass Tag labels to discriminate between treatment group (miR-146a mimic and TLR2 agonist) and control group (scrambled-miR and TLR2 agonist), the effect of miR-146a on the proteome was determined. In total, 172 proteins were identified as being miR-146a regulated and gene ontology assignment resulted in an over-representation of proteins involved in cellular dynamics capable of altering the activation state of microglia. After filtering for bioinformatically predicted targets and those implicated in a similar genomic study, it was decided to further investigate proteins ARF6, RhoA and NOS2 based on their role in modulating the phagocytic potential of microglia. The final objective was to validate miR-146a putative direct targets identified from the proteomics analysis. Luciferase expression of the 3’UTR of targets upon transfection with miR-146a were determined. Based on luciferase analysis, NOS2 appears to be directly targeted by miR-146a and this was also confirmed by western blot. While production of NOS2 by microglia under an acute activation state serves to support and protect CNS homeostasis, chronic expression of this factor can lead to neurotoxicity. Therefore, miR-146a appears to have an overarching role in altering microglial activation during prion disease thus protecting neurons from bystander damage. Taken together, these results suggest that miR-146a could play an important role in the prion disease process by specifically attenuating the microglial induced immune response. Therefore, manipulation of miR-146a may represent a novel therapeutic strategy. Furthermore, given that miR-146a de-regulation has been observed in other neurodegenerative diseases, these results may well extend beyond the realm of prion disease. Lastly, although practical limitations relating to the sensitivity of the comparative proteomics methodology meant that it alone were not sufficient to identify miRNA targets, an integrated approach that takes into consideration genomic and bioinformatic strategies is most promising.

The aim of my thesis was to develop and characterise PrP transgenic Drosophila melanogaster of various genotypes to study the process of prion-induced neurodegeneration in this model. Prion diseases are caused by the occurrence of an abnormally-folded form of PrP (PrPSc) protein that arises either from the environment as an acquired disease, from mutation in the PrP-coding gene as a genetic disease or sporadically from causes unknown. The PrPSc then recruits PrPC, the normal form of PrP, that is ubiquitously present in the mammalian CNS and triggers neurotoxicity and neurodegeneration that is transmissible between individuals of the same or even different species. All prion diseases are currently incurable, fatal and the mechanism of prion-induced neurodegeneration remains to be discovered. In this thesis, Drosophila transgenic for ovine (chromosome 3 and dual PrP transgenic flies), hamster, humanised murine, human and cervid PrP were characterised for expression and biochemical properties. The ultimate goal of my thesis was investigation of cell-to-cell spread of misfolded PrP in Drosophila CNS. To achieve this, a mutant form of PrP that is thought to misfold was co-expressed with the normal form PrPC that served as a substrate in the same dual PrP-transgenic fly. The process was modelled using hamster, humanised murine or ovine PrP transgenes that carry human mutations associated with the spontaneous onset of transmissible neurodegeneration in the natural host. Various approaches towards independent spatial expression of PrP in Drosophila were exploited here in both single and dual PrP expressing flies. Moreover, the ability to initiate misfolding and the impact of this on the fly phenotype was investigated. Both apparent misfolding and phenotypic changes were seen in different fly models suggesting the models were successful. To this extent, PrP transgenic Drosophila were developed to allow for relatively rapid modelling of mammalian prion disease in this invertebrate organism.

<p>Reactions and interactions of iron and oxygen can be both beneficial and detrimental to cells and tissues. Iron is mainly found in our blood where it functions as a mediator in the transport of oxygen to the cells and is further vital for the cellular respiration reducing the oxygen to water. The flexible redox state of iron makes it ideal to contribute in single electron transfers, but may also catalyze reactions with oxygen resulting in cell damaging reactive oxygen species (ROS). Normally the cells are protected against iron toxicity by controlling iron uptake and storage. When the intracellular demand for iron increases; the iron uptake is promoted by increasing the expression of transferrin receptor (TfR) and by decreasing the expression of the iron storage protein ferritin. Ferritin has a central role in the cellular iron detoxification by keeping it in a non reactive but still bioavailable form. However, in neurodegenerative diseases like in Alzheimer’s and Parkinson’s disease the iron storage capacity is disturbed and iron induced oxidative stress adds to the pathology of the diseases. The role of iron and its possible contribution to the pathology of prion diseases, like Creutzfeldt-Jakob disease, is less explored. In the first three studies of this thesis, the iron metabolism and the mutual relation between iron and oxygen are studied in scrapie-infected mouse neuroblastoma cells (ScN2a) as compared to control cells (N2a). In the fourth study we have analyzed the expression of ferritin and TfR in response to inflammation by treating the cells with the bacterial endotoxin lipopolysaccharide (LPS). LPS promotes the expression of inducible nitric oxide synthase (iNOS), a producer of nitric oxide (NO), a well known regulator of the iron metabolism.</p><p>In the first study, the scrapie infection was found to reduce the iron levels, to reduce the mRNA and protein levels of ferritin and the TfR. In addition, reduced levels and activities of the iron regulatory proteins 1 and 2 were observed as compared to the uninfected N2a cells.</p><p>In the second study, the addition of iron to the cell medium strongly increased the level of ROS and decreased the cell viability of the ScN2a cells, whereas the N2a cells were unaffected. The ferritin expression in N2a cells in response to the iron treatment was strongly increased and the concomitant measurement of the labile iron pool (LIP) revealed the LIP to be normalized within four hours. In the ScN2a cells the induction of ferritin expression was lower resulting in elevations in LIP that lasted up to 16 h, indicating that the increased ROS levels were iron catalyzed.</p><p>In the third study, the cells were challenged with hydrogen peroxide (H₂O₂) to elevate the oxidative stress and to analyze the effects on the LIP and cell viability. The ScN2a cells were sensitive to the increased oxidative stress according to the cell viability test, and responded to the treatment with marked increase in the LIP levels, probably derived from an intra-cellular source. The cell viability could be reset by the co-addition of an iron chelator to the cell media. The N2a cells did not elevate the LIP and resisted higher concentrations of H₂O₂ than the ScN2a cells, according to the cell viability assay.</p><p>In the fourth study, the LPS treatment resulted in increased mRNA levels of the heavy chain of ferritin, increased the protein levels of ferritin light chain and decreased the protein levels of the TfR in N2a cells, but no effects were observed in the ScN2a cells. Co-treatment with LPS and the iNOS inhibitor aminoguanidine did not affect the LPS induced decrease of TfR in N2a cells, whereas the free radical scavenger N-acetyl-L-cysteine reversed the effect of LPS on TfR expression, indicating that the changes were mediated by an oxidative rather than a nitric oxide mechanism in the N2a cells.</p>

Reactions and interactions of iron and oxygen can be both beneficial and detrimental to cells and tissues. Iron is mainly found in our blood where it functions as a mediator in the transport of oxygen to the cells and is further vital for the cellular respiration reducing the oxygen to water. The flexible redox state of iron makes it ideal to contribute in single electron transfers, but may also catalyze reactions with oxygen resulting in cell damaging reactive oxygen species (ROS). Normally the cells are protected against iron toxicity by controlling iron uptake and storage. When the intracellular demand for iron increases; the iron uptake is promoted by increasing the expression of transferrin receptor (TfR) and by decreasing the expression of the iron storage protein ferritin. Ferritin has a central role in the cellular iron detoxification by keeping it in a non reactive but still bioavailable form. However, in neurodegenerative diseases like in Alzheimer’s and Parkinson’s disease the iron storage capacity is disturbed and iron induced oxidative stress adds to the pathology of the diseases. The role of iron and its possible contribution to the pathology of prion diseases, like Creutzfeldt-Jakob disease, is less explored. In the first three studies of this thesis, the iron metabolism and the mutual relation between iron and oxygen are studied in scrapie-infected mouse neuroblastoma cells (ScN2a) as compared to control cells (N2a). In the fourth study we have analyzed the expression of ferritin and TfR in response to inflammation by treating the cells with the bacterial endotoxin lipopolysaccharide (LPS). LPS promotes the expression of inducible nitric oxide synthase (iNOS), a producer of nitric oxide (NO), a well known regulator of the iron metabolism. In the first study, the scrapie infection was found to reduce the iron levels, to reduce the mRNA and protein levels of ferritin and the TfR. In addition, reduced levels and activities of the iron regulatory proteins 1 and 2 were observed as compared to the uninfected N2a cells. In the second study, the addition of iron to the cell medium strongly increased the level of ROS and decreased the cell viability of the ScN2a cells, whereas the N2a cells were unaffected. The ferritin expression in N2a cells in response to the iron treatment was strongly increased and the concomitant measurement of the labile iron pool (LIP) revealed the LIP to be normalized within four hours. In the ScN2a cells the induction of ferritin expression was lower resulting in elevations in LIP that lasted up to 16 h, indicating that the increased ROS levels were iron catalyzed. In the third study, the cells were challenged with hydrogen peroxide (H2O2) to elevate the oxidative stress and to analyze the effects on the LIP and cell viability. The ScN2a cells were sensitive to the increased oxidative stress according to the cell viability test, and responded to the treatment with marked increase in the LIP levels, probably derived from an intra-cellular source. The cell viability could be reset by the co-addition of an iron chelator to the cell media. The N2a cells did not elevate the LIP and resisted higher concentrations of H2O2 than the ScN2a cells, according to the cell viability assay. In the fourth study, the LPS treatment resulted in increased mRNA levels of the heavy chain of ferritin, increased the protein levels of ferritin light chain and decreased the protein levels of the TfR in N2a cells, but no effects were observed in the ScN2a cells. Co-treatment with LPS and the iNOS inhibitor aminoguanidine did not affect the LPS induced decrease of TfR in N2a cells, whereas the free radical scavenger N-acetyl-L-cysteine reversed the effect of LPS on TfR expression, indicating that the changes were mediated by an oxidative rather than a nitric oxide mechanism in the N2a cells.

Prion diseases are neurodegenerative disorders that can affect humans and animals. The underlying event is a conformational change of the normal cellular prion protein (PrPC) into an aberrant isoform termed PrP-scrapie (PrPSc). PrPSc is thought to lead to neurodegeneration and activation of glial cells. Scrapie infection of neuroblastoma cells was shown to increase the expression of insulin receptor (IR). Additionally, a marked reduction of 125I-insulin binding sites was observed. Insulin stimulation showed alteration in both IR β-subunit tyrosine phosphorylation and extracellular signal regulated kinase-2 (ERK2) activity.  Furthermore, scrapie infection was shown to increase insulin-like growth factor-1(IGF-1) receptor (IGF-1R) expression, although the number of 125I-IGF-1-binding sites was reduced. Also binding affinity of 125I-IGF-1 to its receptor was reduced, and tyrosine phosphorylation of IGF-1R-β-subunit in response to IGF-1 was altered. The increased levels of neurotrophic receptors might represent a neuroprotective response to prion infection. However, scrapie infection instead leads to decreased function, decreased levels of functional receptors, or both, which could promote neurodegeneration in prion diseases, through attenuated neurotrophic support. In BV-2 microglial cells, LPS-induced iNOS (inducible nitric oxide synthase) expression and subsequent NO production were mainly mediated through c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathway. Antioxidant treatment indicates that oxidative suppressing mechanism(s) acts on JNK pathway possibly as a regulatory mechanism controlling the NO levels. The JNK pathway was also shown to play an important role in the survival of BV-2 cells. We show that BV-2 cells are protected from ongoing apoptosis by pro-survival activity mediated both by the JNK and p38 MAPK pathway during LPS-induced inflammation. This is very interesting findings since it is important for microglia to respond properly to a pathogen, without themselves being affected and undergo apoptosis.

No description available.

No description available.

The Transmissible Spongiform Encephalopathies (TSEs) or “prion” diseases are a group of fatal neurodegenerative diseases the aetiology of which is not fully understood. These diseases are characterised by a number of pathological changes in the central nervous system (CNS) including; vacuolation of the neuropil, gliosis and deposition of PrPSc; the abnormal form of the host glycoprotein PrP. Although the major pathology in these diseases is associated with the CNS the immune system is central to the pathogenesis of many natural and experimental TSEs including natural scrapie in sheep, chronic wasting disease in free ranging and captive deer and variant CJD (vCJD) in humans. Unlike many infectious diseases where deficiencies in immune function are opportunistic for the invading pathogen a competent immune system is required for efficient TSE infection via peripheral routes. As infection of the lymphoid tissues in many TSEs can occur many months before the detection of infectivity in the CNS, the determination of those cells in the lymphoid system has been the focus of much research and a number of studies now point towards the importance of the follicular dendritic cell (FDC), a long-lived radio resistant cell, in TSE pathogenesis. The involvement of FDCs in peripheral TSE pathogenesis relates to the inability of ionising radiation to influence pathogenesis, the association of PrP protein with FDCs in both uninfected and infected lymphoid tissues, and the demonstration that TSE pathogenesis is severely impaired in mice devoid of these cells. The aims of this thesis were to further understand the role of FDCs in the pathogenesis of a range of mouse-adapted experimental TSE strains and to determine if peripherally acquired TSE infections are influenced by host age or by stimulation of the immune system. Using chimaeric mouse models where a mismatch in the expression of PrP protein between FDCs and lymphoid/myeloid cells was produced, further evidence for a critical role for in the pathogenesis of the ME7 TSE strain was produced. Although these findings produced strong evidence that FDCs were important for the ME7 strain the possibility that different TSE strains may target different cell types in the peripheral lymphoid system was explored using a range of mice with specific immunological defects. Infection of these mice with several experimental TSE strains showed that the presence of mature FDCs was also important for the pathogenesis of the strains tested. Clinical cases of vCJD have been confined almost exclusively to young adults, although the reasons behind this apparent age-related susceptibility are not fully understood. The capacity of the immune system to mediate immune responses to pathogens declines with age as a result of impaired lymphocyte and FDC function. As FDCs are critically involved in the pathogenesis of many TSEs, including vCJD, it was hypothesised that an aging immune system may impair disease pathogenesis. Peripheral infection of senescent mice failed to produce clinical disease during lifespan, although evidence of disease transmission, was detected in a proportion of aged mice. These findings demonstrate that this inefficient disease transmission, as a consequence of age, may lead to considerable levels of sub-clinical disease within the population. Finally the influence of immune system stimulation, by the generation of a humoral immune response, on peripheral TSE pathogenesis was investigated. These findings demonstrated that immunisation can influence pathogenesis, but only during the early stages of infection prior to spread to the CNS. These data imply that modulation of the immune system does not alter TSE pathogenesis once disease has been initiated in the CNS. Finally, these studies have found some preliminary evidence that TSE infection may induce FDC activation suggesting that TSE infection may influence the immune response. Together, these data show that a functional immune system and specifically, the presence of mature FDCs, are central to the pathogenesis of peripherally acquired TSE infections.

Pathogenesis of prion diseases in animals is associated with the misfolding of the cellular prion protein PrPC to the infectious form, PrPSc. We hypothesized that an animal’s susceptibility to prions is correlated with the propensity of an animal’s PrPC to adopt a β-sheet, PrPSc-like, conformation. We have developed a method which uses circular dichroism (CD) to directly calculate the relative population of PrP molecules that adopt a β-sheet conformation or the ‘β-state’, as a function of denaturant concentration and pH. We find that the PrP from animals that are more susceptible to prion diseases, like hamsters and mice, adopt the β-state more readily than the PrP from rabbits. The X-ray crystal structure of rabbit PrP reveals a helix-capping motif that may lower the propensity to form the β-state. PrP in the β-state contains both monomeric and octameric β-structured species, and possesses cytotoxic properties.