1,720,975 research outputs found
In vitro and in vivo evaluation of listeria monocytogenes based protein and DNA delivery
No full textListeria monocytogenes was chosen as a carrier for vaccine delivery to take advantage of several unique features. It is capable of invading, surviving and replicating in the majority of mammalian host cells. Due to its abilities to infect phagocytic and non-phagocytic cells and to survive in the cytoplasm of mammalian cells, it is a promising vector for oral delivery of vaccines, providing that safety measures can be built into the system to reduce its pathogenicity. Since the organism is capable of infecting the gastrointestinal epithelium, it is particularly attractive as a basis for designing a generic delivery system for oral vaccination. In this study, a stable recombinant suicidal strain of L. monocytogenes (rsΔ2), developed previously in our laboratory, was used for protein and DNA delivery. The L. monocytogenes strain (rsΔ2) contains the cell wall hydrolysis gene “ply118” and its associated holin gene from a Listeria specific phage A118. These genes are integrated into the L. monocytogenes genome and promote lysis of the rsΔ2 strain when the bacterial cells reach the cytoplasm of mammalian cells. The engineered plasmid pDuLX is a dual expression vector that can drive gene expression from each of two promoters, the Listeria promoter (phly) and the commonly used eukaryotic viral promoter (PCMV). An early objective of this study was to use the luciferase reporter gene to demonstrate that luciferase protein can be detected after uptake of the rsΔ2 strain containing pDuLX-Luc by Caco-2 cells. The luciferase reporter demonstrated that pDuLX can deliver protein and DNA to dividing Caco-2 human epithelial monolayers. Non-dividing (fully differentiated) cells were resistant to lipofectamine transfection, a finding that can be explained by lack of DNA delivery to the nucleus. It was demonstrated that when differentiated Caco-2 monolayers were treated with rsΔ2, the bacteria were able to deliver a significant quantity of luciferase protein produced by the Listeria cells. By implication the bacteria were also able to deliver DNA, but expression driven by the eukaryotic promoter in differentiated Caco-2 cells was not observed. The presence of luciferase in non-dividing Caco-2 cells was explained by activation of the promoter Phly in pDuLX which drove expression of luciferase when the bacteria were grown in charcoal-treated BHI medium. This results in presence of reporter protein in the Listeria cells prior to delivery into the host Caco-2 cells. Eukaryotic expression of luciferase in non-dividing cells was absent because the pDuLX plasmid lacked a mechanism to enter the nucleus. When the rsΔ2 strain was taken up by Caco-2 cells, there was little or no bacterial growth, whereas when the control Δ2 strain was taken up it was shown to be viable and multiplied by approximately three log cycles over a 2-day period within the Caco-2 cells. A small mass of protein or DNA also appeared to be delivered to host cells by the Δ2 strain, perhaps because some bacteria died but, despite the extent of bacterial growth, the mass of protein delivered to dividing Caco-2 cells by the Δ2 strain was considerably less than that delivered by the rsΔ2 strain. In vivo experiments to test the rsΔ2 strain as a potential delivery system for vaccines were carried out in a mouse model using ovalbumin as the test vaccine via either intramuscular or oral administration. After intramuscular injection, the bacterial strain, rs∆2(pDuLX-OVA) produced significantly higher titres of antibody and was also more effective at inducing targeted T cell lysis, when compared to negative controls and other strains. This could be explained by delivery of OVA originally expressed from the prokaryotic promoter present in pDuLX-OVA. Some activity was evident using rs∆2(p3L-OVA) which does not contain the prokaryotic promoter, suggesting that some transfection of mouse myocytes, other cells present in muscle, or infiltrating cells of the immune system, occurred after intramuscular injection. Delivery of the negative control rs∆2(pDuLX-Luc) confirmed that the observed activity was induced specifically by the ovalbumin vaccination. Tests of oral ovalbumin vaccination of mice were performed by gavage using rs∆2(pDuLX-OVA) and the negative control rs∆2(pDuLX-Luc). After a single dose there was evidence that the system was also effective as an oral vaccine. Further work will be required to investigate biological activity after multiple oral doses to optimise a dosage regimen for oral vaccination
In-vitro evaluation of factors that affect the performance of lipid-based drug delivery systems
No full textDevelopment of formulations containing poorly water-soluble drugs (PWSDS) incorporated in lipid-based drug delivery systems (LBDDS) poses a great challenge to scientists (both, at academia and industry) across the globe. To date there are no standard in vitro protocols for formulation scientists which predict their performance in-vivo. This thesis addresses various key issues that are important in development of LBDDS. Chapter 1 sets the work in context by reviewing the published literature. Work carried out in Chapter 2: addressed the issue of "non-completion of lipolysis" as this has been attributed by many authors to be one factor that limits the use of in vitro digestion tests, and limits the degree of in vitro- in vivo correlation (IVIVC) that can be achieved. This study has investigated the effect of increasing calcium and bile salt (BS) concentration on the in vitro digestion of a long-chain triglyceride (soybean oil) in order to understand how these factors will affect the solubility of poorly water-soluble drugs delivered in lipid vehicles. The solubility of two model poorly-water soluble drugs (fenofibrate and Danazol ) in the aqueous phase digests obtained via digestion of a long-chain triglyceride, LCT (soybean oil), increased significantly in each of the conditions (fasting and fed), by comparison with respective controls, irrespective of the molar concentration of calcium employed in the media. Systems containing 40 mM calcium concentration (high levels of calcium used in the study) when compared to that containing 5 mM calcium, had a lower capacity for solubilization of either drug in aqueous phases after digestion, in both fed and fasting conditions. This was thought to be attributed to the formation of large amount of insoluble calcium soaps which were observed (as a precipitate) during our experiments. Formation of calcium soaps has been reported elsewhere (MacGregor et al., 1997, Hu et al., 2010, Zangenberg et al., 2001a). Soap formation may occur upon an interaction of calcium with the bile salt component of the solubilized species (Fatouros et al., 2009) when calcium is in excess. In conclusion, from our data, although high calcium concentration may prove beneficial with respect to bringing the lipolysis to completion, the addition of calcium ions should be conducted with caution because it interferes with the solubilisation of poorly water soluble drugs. Therefore, it can be anticpated that high concentrations of calcium in the system during in vitro lipolysis will result in a poor model for correlation in vivo. A second focus of this thesis is discussed in Chapter 3: describes an investigation of a series of closely related SEDDS viz. Type II and Type IIIA as defined by the Lipid Formulation Classification System (LFCS) (Pouton, 2006b, Pouton, 2000b), all of which contained fenofibrate as model drug. A variety of factors influencing the performance of these systems during in vitro dispersion and digestion tests were studied. The results were interpreted based on the level/extent of supersaturation attained during these in vitro processes to gain an insight into formulation performance and to establish guidelines for formulators. Emphasis was placed on the effects of lipid composition (long-chain vs. medium-chain) and the surfactant type (hydrophilic vs. lipophilic) on the solubilization properties of these formulations during dispersion and digestion. Despite generating diverse formulations by altering the nature of oils and blends of oils which made up the lipid component, the dispersion results showed that Type II formulations (containing Tween 85, a lipophilic surfactant) always supported drug in solubilized form (100%) for at least 4 days (in the absence of digestion), Type III formulations on the other hand were unable to maintain all of the drug in solubilized form on dispersion, though they maintained greater than 70% of drug in solubilized form for 4 days. Most of the loss of drug in the form of precipitate occurred after the initial 4 hours.. The degree of supersaturation generated during dispersion was estimated by determining the solubility of fenofibrate in dispersed formulations. Type III formulations were supersaturated and drug was maintained in this meta-stable state for up to 4 hours and after which drug was lost to some extent in the form of precipitate. Type II systems were not supersaturated. Considering the transit time of all the formulations in the intestine was expected to be 3-4 hours, clearly Type II and Type IIIA formulations, prior to digestion, met the primary performance requirement for drugs meant to be administered orally. After dynamic digestion studies, the ability of each of these formulations (Type II and Type IIIA) to maintain drug in a solubilized state was highly dependent on both, the lipid composition and the choice of surfactant. For example, medium-chain lipids exhibited very good solubilizing properties in the dispersed state, but resulted in a higher degree of supersaturation on digestion, leading to higher susceptibility to drug precipitation. Results from the digestion studies showed that replacing long-chain lipids with medium-chain lipids in Type II and IIIA LBDDS is likely to promote supersaturation on digestion. Utilization of long-chain instead of medium-chain triglycerides in LBDDS prevents the development of sudden and higher degrees of supersaturation and consequently reduces the risk of precipitation (Kossena et al., 2003a). The present digestion studies in Chapter 3: have indicated that this approach alone will not work for all drugs. For fenofibrate, various other strategies needs to be explored to prevent drug precipitation from formulations, such as lowering the drug load (Williams et al., 2012a), employing polymer-based precipitation inhibitors (Anby et al., 2012c), and/or by the careful selection of surfactants (Cuine et al., 2008a). Without careful consideration of drug loading and choice of surfactant in Type II/IIIA medium-chain lipid formulations, there is a high risk of precipitation of drug in the intestine. Critical to the utility of self-emulsifying drug delivery systems (SEDDS) in oral bioavailability enhancement is a capacity to both generate and maintain supersaturation following dispersion and digestion processes in the gastro-intestinal tract. Studies carried out in Chapter 4: investigated the effect of drug-type and drug loading on supersaturation in digested SEDDS consisting of long-chain lipids and a range of chemically diverse nonionic surfactants. Supersaturation is described in terms of the maximum supersaturation ratio (SRM) attained on initiation of digestion. Calculated from the maximum attainable concentration in the test (a function of drug loading) and the drug solubility in the colloidal phases formed by digestion of the SEDDS, SRM defines the maximum supersaturation pressure in the digestion experiment and proves to be a remarkable indicator of performance across a range of formulations. SEDDS containing danazol showed little evidence of precipitation on digestion, even at drug loads approaching saturation in the formulation. In contrast, fenofibrate extensively crystallized on digestion of the same SEDDS. The performance differential of danazol and fenofibrate-containing SEDDS however could be rationalized by the much higher SRM values generated by fenofibrate. And on further analysis of formulations containing various fenofibrate loads, a threshold SRM of ~2.6 was identified in 6 of the 7 SEDDS above which supersaturation could not be maintained. Near this threshold, performance became increasingly variable and most sensitive to surfactant-type, though overall, the SRM attained on digestion was most predictive of performance
Generation of mouse embryonic stem cell reporter lines to identify developmental stages during induction of dopaminergic neurons
No full textThe most fundamental questions on how neurons arise from specific developmental programs focus on how they acquire their correct synaptic connections, and how they mature and develop their distinct phenotypes. It is known in a broad sense that each group of neurons in the functional circuits of the adult brain develops according to both intrinsic programming of molecular cascades, and extrinsic influences from their environments. Here I set out to investigate the cells of the rostral region of the central nervous system, those of the mesencephalon, or midbrain, by the use of murine embryonic stem (ES) cells as an in vitro model of midbrain dopaminergic (mDA) progenitor cells. The focus of the work was to introduce genetic reporters into ES cells to allow identification and potentially isolation of neuronal progenitors during in vitro differentiation of ES cells. mDA neurons arise in the ventral midline of the midbrain, and are intrinsic in such functions as fine motor control, emotion, perception and higher cognition, as well as learning and reward. Loss of these neurons in the substantia nigra leads to the symptoms of Parkinson's disease (PD). Before ES cell therapies can attempt to treat PD, the signalling factors involved in the early specification of mDA neurons need to be thoroughly characterised. Differentiation of ES cells using existing protocols does not result in homogeneous populations of neural progenitors. Typically, mouse ES cells differentiate into neural progenitors by default once ES cell growth supplements are removed, but rarely are these mDA progenitor neurons. In this thesis, the neural induction protocol was first investigated to examine whether it was generating neural progenitors of a midbrain phenotype using an Lmx1a-eGFP reporter cell line, as a study control. The induction protocol was found to generate, at its peak, 8.1% of the total cell population expressing the fluorescent reporter. This indicated that the induction protocol was not yielding enriched populations of Lmx1a+ neural progenitors. Additionally it was considered that it may be that not all of the Lmx1a-eGFP+ cells were destined to become dopaminergic neurons, and that perhaps more reporters were needed to identify midbrain dopaminergic neurons. Following this, a series of homologous recombination experiments were performed, initially successfully producing an Lmx1b-CBR-IRES-dsRed reporter in a murine ES (mES) cell line, with 17.9% targeting efficiency. Following this a series of targeting experiments were carried out in an attempt to target a Pitx3-mCitrine reporter to an existing Lmx1a-AMP mES reporter cell line. Unfortunately, despite the expansion and screening of over 1000 antibiotic resistant colonies over a 15 month period, this targeting campaign was not successful. In an effort to assess the cause for this lack of success, an additional targeting experiment was successfully performed, using an Lmx1a-mCFP targeting vector to recombine with a wild-type mES cell line, with a 3.45% targeting efficiency. In all likelihood the failure of the Pitx3-mCitrine to target adequately lies with the design of the targeting vector and primers used; the homology of the vector to host genome was 7kb in total, which may in fact have been in excess when considering that the target, Pitx3, is expressed late in the differentiation of DA neurons, giving rise to the possibility that the DNA is tightly bound and comparatively inaccessible at this undifferentiated state. The intention for the generation of dual Lmx1a-postive/Pitx3-positive fluorescent reporter cell lines was to enable the identification of DA neuronal progenitors, with the goal of ultimately using these cells to isolate DA cells at definite specific stages of development prior to transplantation into a mouse model of PD. Although the Pitx3-mCitrine targeting did not succeed, the neural induction protocol was successfully assayed using an Lmx1a-eGFP reporter cell line, a novel Lmx1a-mCFP reporter cell line was created, as was a novel Lmx1b-CBR-dsRed cell line
Molecular dynamics simulations of lipid-based drug delivery systems
No full textMolecular dynamics (MD) simulation is a powerful technique to investigate molecular self-assembly. It can be used to model and understand the interactions of biological membranes, proteins, and lipids. Above their critical micelle concentration (CMC), molecules that are composed of hydrophilic head group and hydrophobic tail group aggregate spontaneously to form a wide variety of assemblies ranging from micelles, rodlike structures, and bilayers to more complex phases such as hexagonal and cubic phases. These self-assembly processes are of fundamental importance in drug discovery and development. In the area of drug discovery and development, it is vital to have an effective means of improving the bioavailability of poorly water-soluble drugs (PWSD). Lipid-based delivery systems (LBDDS) are one of the important approaches of improving the bioavailability of PWSD. The nature of gastrointestinal (GI) fluids strongly influences the absorption of PWSDs. The dissolution rate and the amount of drugs dissolved is determined by the nature of the GI fluids and their solubilisation capacity. Within the GI tract there are endogenous as well as exogenous solubilising components. The endogenous components are secreted from the gall bladder, whereas the exogenous components are those which are administered in the drug formulation as well as resulting from meals. After oral administration, drugs must remain dissolved within the GI tract before partitioning into and then across the enterocyte. Although the self–assembly process of lipids and lipophilic excipients within the GI tract are thought to have a significant influence on drug solubilisation and the degree of drug supersaturation, the molecular understanding of these structures is limited. The first section of this work describes the modification of the GROMOS 53A6 united atom force field particularly for polyethylene glycol (PEG). Then, using MD simulations and experimental methods such as turbidity, particle size measurement, cross-polarized light microscopy and NMR, the current study explores the phase behaviour of (i) the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sodium glycochenodeoxycholate (GDX), and water system, and (ii) the 1-palmitoyl-2-hydroxy-sn-glycerol-3-phosphocoline (Lyso PC), GDX and water system and constructs ternary phase diagrams of these mixtures. It also investigates part of the quaternary phase diagram of Lyso PC, glycerol 1-monooleate (GMO), GDX and water, which was used to investigate the structures formed in the intestine after digestion of triglycerides. The solubilisation capacity of the lipidic microenvironment on PWSD has also been investigated using LC-MS and MD simulation. The association structures of these various systems have been modelled and compared to the experimental phase behaviour of the analogous systems. It is indicated in these studies that digestion and digested products have a significant impact on the phase behaviour of the contents of the small intestine and on solubilisation and bioavailability of PWSDs. In summary, this thesis contributes to a better understanding of the performance of lipid-based formulations (LBF) and shines a light on the use of MD simulations as a prediction tool to model LBDDS
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Haplotyping with peptide nucleic acids: a novel method for tissue typing
No full textIn the effort to become the first to establish the “thousand dollar genome,” a fundamental component has been outside the main focus of many geneticists: the appreciation of phase. For any genotype or allele, flanking elements can have varying degrees of influence, which limits the effectiveness of multifactorial trait/disorder mapping. Several types of laboratory means can resolve unambiguous sequences up to several hundred thousand bases in length however the longer the sequence generally means increases in time, labour and expense. This project sought to develop a means of providing a simple, robust and effective procedure to target and isolate specific DNA sequences in order to enable the unambiguous sequence. The target was decided as the human leukocyte antigen system, a cluster of genes with a well-established pattern of allelic linkage. In order to target this region, a new class of molecule, peptide nucleic acids, were bound to nominated alleles selected. The peptide nucleic acids were designed with fluorescent and biotin moieties to enable their detection and for solid phase extraction (i.e. magnetic beads/microplate) once bound to their matching allele. A probe-targetextraction methodology was used as a template for basing the protocol. To facilitate the development of the process to use on genomic DNA, two allelespecific plasmid constructs were generated to test the process under favourable conditions. Increases in the targeted allele were detected by allele specific quantification using real-time polymerase chain reaction and sequencing. The assay was adapted for use with genomic DNA, extracted from whole blood samples with a custom extraction protocol designed to maximise the molecular weight of the DNA. The assay was tested for a range of variables aimed at determining factors that influence the allelic ratio pre and post assay. Optimising the hybridisation conditions, starting amount, probe to target ratio and wash number influenced the degree of allelic separation. Further experimental work is required on yielding more consistent results in order to reliably produce haploid DNA sequences
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Evaluation of the role of leukaemia inhibitory factor receptor antagonist in the pathogenesis of inflammatory arthritis
No full textRheumatoid arthritis (RA) is a chronic, inflammatory, systemic, autoimmune disease that is characterized by acute inflammation of the synovium, cartilage destruction and bone damage. The exact cause of the disease has not been elucidated so far. Nevertheless, the pathogenesis of RA is quite complex, involving a repertoire of cytokines that act either collectively or individually to exert their pro-inflammatory effects, thereby debilitating the joint architecture. Despite the success of several anti-cytokine based therapies, particularly those targeting tumour necrosis factor (TNFα), in the treatment of rheumatoid arthritis (RA), there is still a need for development of alternative therapies to treat RA patients, who are either refractory to the existing therapies or, who discontinue the use of these drugs owing to their potential side effects and/or toxicities. Leukaemia inhibitory factor (LIF) is an IL-6 subfamily cytokine that has been well implicated in the pathogenesis of RA. Considering this, the development of anti-LIF therapeutics as an alternative to the currently available treatments in RA is a promising possibility that merits further study. Several LIF antagonists have been developed to block the biological activity of LIF. However, whether antagonism of LIF and the other related IL-6 subfamily cytokines would favourably influence the disease severity or outcome in RA has not been fully investigated so far. This study aimed at evaluating the role of a LIF receptor super antagonist, MH35BD, in the pathogenesis of inflammatory arthritis. MH35BD was observed to be exerting anti-inflammatory and chondroprotective effects in vitro. In addition, it was found to block the biological activity of other IL-6 subfamily cytokines, such as Oncostatin M (OSM), also implicated in the pathogenesis of RA. Antagonsim of LIFR by MH35BD reduced both LIF and OSM induced expression of proteolytic endopeptidases and their naturally occurring inhibitors by primary articular chondrocytes to basal levels. Furthermore, MH35BD had no effect on STAT3 phosphorylation in murine macrophages, thereby potentially blocking the gp130 receptor subunit-mediated signalling via the JAK/STAT pathway in macrophages. With the aim of increasing the serum half-life of MH3BD, the MH35BD:Fc fusion protein construct was generated and tested in vivo for its serum stability. However, the MH35BD:Fc fusion protein was found to be less stable in the serum as compared to its monomeric counterpart, MH35BD, since the serum levels of MH35BD:Fc decreased at a rapid rate than that of MH35BD. This highlights the importance of studying the binding characteristics of Fc based fusion proteins to neonatal Fc receptors, FcRn that determines their serum stability. When tested for it efficacy in animal models of arthritis, MH35BD did not exert any significant effect on the extent of joint inflammation in both K/BxN serum induced and antigen induced (AIA) models of arthritis. Nevertheless, it significantly inhibited the cutaneous delayed type hypersensitivity (DTH) reactions induced in arthritic mice, suggesting that the T cell initiated joint inflammation in AIA and DTH reactions may be regulated by independent mechanisms. The very aggressive nature of the disease in K/BxN serum induced model of arthritis and the issues pertaining to the completeness of the blockade of LIFR by MH35BD might have been responsible for the ineffectiveness of the anti-LIF therapy in this model of inflammatory arthritis. However, these issues were circumvented by inducing arthritis in LIF gene knockouts (LIF-/-) by K/BxN serum transfer. Complete absence of endogenous LIF production in the LIF knockouts ensured proper interpretation of the observed results, without any interference in respect to parameters such as sufficiency of dose and completeness of blockade of LIFR. It was found that although the LIF-/- mice were susceptible to arthritis induction by K/BxN serum transfer, the disease severity in LIF-/- mice was substantially ameliorated as compared to the wild-type diseased controls. In conclusion, this study provides a useful insight into the role of LIFR antagonist, MH35BD, as a potential therapeutic candidate for the treatment of rheumatoid arthritis and other related arthropathies. Compelling evidence has proved that LIF contributes to the induction and progression of inflammatory arthritis and joint tissue damage in the K/BxN serum induced arthritis. Thus, this study provides further impetus to test the effects of LIF blockade as a potential therapeutic strategy in rheumatoid arthritis
Investigating the development of midbrain dopaminergic neurons using mouse embryonic stem cell reporter lines
No full textEmbryonic stem (ES) cells possess the capability to self-renew indefinitely and are capable of generating any cell of the three primary germ layers, making them an attractive source of material to investigate both basic physiological properties and neurodegenerative processes. Although ES cells can be directed into specific cell lineages, the differentiation of ES cells results in heterogenous cultures. To date, there are few differentiation protocols that produce homogenous populations of any desired cell type. Many methods have been used in an effort to obtain homogenous populations of cells; from forced expression of genes involved in developmental pathways, to FACS isolation of cells expressing markers of interest. There has been a considerable focus on generating homogenous populations of midbrain dopaminergic progenitors (or neurons) for Parkinson's disease which involves the degeneration of a specific population of midbrain dopaminergic neurons. In this thesis, I investigate the development of mouse embryonic stem cells into midbrain dopaminergic neurons using reporter cell lines. In the first experimental chapter, I investigate the expression of Lmx1a and Msx1; two key transcription factors implicated in dopaminergic neuronal development. I also examine the impact of the BMP, Shh and Wnt signalling pathways on dopaminergic neural differentiation. Activation of the BMP and Wnt pathways resulted in inhibition of neural induction and the expression of both Lmx1a and Msx1. In contrast, antagonising these signalling pathways increased the yield of tyrosine hydroxylase (TH) expressing neurons. Activating or inhibiting the Shh pathway did not affect Lmx1a, Msx1 or TH expression. These experiments show that early Lmx1a expression is not indicative of the number of dopaminergic neurons produced. Furthermore, many of the TH positive neurons derived from monolayer cultures were not of midbrain origin. In the following experimental chapter, I used immunocytochemistry and qPCR to characterise the population of cells expressing Lmx1a. The downstream targets of Lmx1a, Msx1 and Wnt1, and midbrain dopaminergic neuron markers, Lmx1b and En1, were significantly upregulated in Lmx1a positive cells. The Lmx1a positive fraction was enriched with neural progenitors, and give rise to highly neural cultures. However, the majority of neurons in the terminally differentiated cultures derived from Lmx1a positive cells were GABAergic. Immunocytochemistry identified these cells as forebrain GABAergic neurons with upper-layer identity. Furthermore, the isolated Lmx1a positive cells were not responsive to patterning cues, indicating that they were already committed towards a GABAergic neuron fate. To show that these Lmx1a+ progenitors could generate dopaminergic neurons I used an alternative differentiation paradigm, the PA6 co-culture method. Expression of Lmx1a in PA6 co-cultures was different from monolayer cultures; the percentage of Lmx1a positive cells increased throughout the differentiation period. In addition, PA6 co-culture derived TH positive cells were found to co-express Lmx1a, an occurrence that was uncommon in monolayer cultures. The ionotropic glutamate receptors on neurons derived on adherent monolayer and PA6 co-cultures were functionally characterised in the final experimental chapter. Previously, antagonism of ionotropic glutamate receptors has been reported to improve behavioural assay scores in Parkinsonian animal models (Johnson et al., 2009). Terminally differentiated monolayer cultures and PA6 co-cultures responded differently to stimulation with glutamate, AMPA kainate and NMDA. The ionotropic glutamate receptors of midbrain dopaminergic and GABAergic neurons derived from both culture systems were further investigated. An initial characterisation indicates distinct differences between the glutamate receptor populations in monolayer and PA6 co-cultures. It appears that monolayer differentiation generates AMPA expressing midbrain dopaminergic neurons in comparison to the NMDA receptors evident following PA6 differentiation. Interestingly, these differences in receptor expression appear restricted by culture method, rather than neuronal subtype, i.e. monolayer neurons expressed AMPA receptors, regardless of whether they were TH+ or GAD67+. Similarly both TH+ and GAD67+ neurons appeared to express NMDA receptors following PA6 differentiation. At present the significance of these findings is unknown. In addition, the effect of Wnt5a on cell responses to glutamate agonists was examined. Wnt5a was able to potentiate cell responses to sub-maximal concentrations of certain glutamate agonists depending on the differentiation paradigm performed
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