Neural Stem Cell Grafts and the Influence of Apolipoprotein E in a Mouse Model of Global Ischaemia

Neural stem cell (NSC) transplantation is a promising therapy for the treatment of brain damage. Although the “proof of principle” for NSC transplantation therapy has been demonstrated in a variety of animal models of brain injury (stroke, traumatic brain injury, ageing) and in a clinical setting (Parkinson’s disease), the mechanisms by which grafted stem cells survive, migrate and differentiate in host brain are yet to be elucidated. Initial studies have demonstrated that, after transplantation of the MHP36 neural stem cell line in a focal ischaemia model, the lipid transport protein apolipoprotein E (apoE) is upregulated and co-localised to differentiated cells in parallel with functional recovery. ApoE has been shown to have a critical role in the response to brain injury and repair processes. Furthermore, in humans, three different forms of apoE exist (E2, E3, E4 encoded by the alleles e2, e3, e4) and each of these has a different ability to promote repair, with the E4 form associated with an impaired capacity. This thesis tests the hypothesis that apoE is critical in stem cell integration and investigates whether this effect is APOE genotype dependent, in a mouse model of global cerebral ischaemia. This model was chosen as it produces diffuse selective neuronal damage in the striatum and hippocampus, which also occurs in other conditions such as ageing and Alzheimer’s disease. The studies described in this thesis were designed to test the hypothesis and are outlined as follows: I. Characterisation of neural stem cell grafts in a mouse model of global ischaemia In order to investigate the potential influence of apoE on stem cell grafts, it was first essential to characterise stem cells grafts in mouse brain. Thus, the initial aim of the thesis was to characterise MHP36 grafts in a mouse model of ischaemic neuronal injury. The effect of cyclosporin A (CsA) immunosuppression was also investigated. C57Bl/6J mice underwent an episode of transient global ischaemia induced by bilateral common carotid artery occlusion. Three days following ischaemia, mice received a unilateral striatal graft of fluorescently labelled MHP36 neural stem cells or vehicle; the mice also received CsA or saline. The mice were terminated at either XVII 1 or 4 weeks post-transplantation. This study determined that MHP36 grafts survived and migrated robustly in host ischaemic brain at both 1 week and 4 weeks post-transplantation. Grafted MHP36 cells differentiated into neurons and were able to reduce the extent of ischaemic neuronal damage. An acute host inflammatory response was evoked following MHP36 grafting, but this decreased dramatically by 4 weeks post-transplantation. CsA immunosuppression did not affect MHP36 survival and migration or reduce the host inflammatory response. The successful transplantation and characterisation of MHP36 grafts in mouse brain allowed for future investigation into the genetic factors underlying stem cell graft integration via the use of apoE transgenic mice. II. Influence of apoE on neural stem cell grafts in a mouse model of global ischaemia The aim of this study was to investigate whether endogenous apoE influenced MHP36 survival, migration and differentiation and then to determine potential signalling pathways that may be involved. ApoE deficient mice on a C57Bl/6J background (APOE-KO) and control wildtype C57Bl/6J (WT) mice were subjected to an episode of transient global ischaemia, as in Experiment 1. Two weeks following ischaemia, all mice received unilateral striatal and hippocampal grafts of MHP36 cells. All mice received CsA immunosuppression. Mice were terminated 4 weeks post-transplantation. MHP36 survival and migration was significantly increased in WT as compared to APOE-KO mice. In addition, neuronal differentiation was significantly increased in WT as compared to APOE-KO mice. Increased astrocytic differentiation was observed in the hippocampus, but not striatum of WT as compared to APOE-KO mice. Measurement of the levels of signalling proteins associated with cell survival, extracellular signal-regulated kinase (ERKs) and c-Jun amino-terminal kinase (JNKs) and their phosphorylated forms (pERK and pJNK), indicated selective alterations in JNK with no change in ERK in APOE-KO as compared to WT mice, suggesting that JNK may underlie the apoE effects in stem cell integration. This study demonstrated that apoE strongly influences the survival, migration and differentiation of grafted MHP36 cells and provides initial evidence for the signalling pathways involved. XVIII III. Influence of APOE genotype on neural stem cell grafts in a mouse model of global ischaemia Following the demonstration that endogenous mouse apoE has a critical role in MHP36 graft survival, migration and differentiation, this study sought to investigate whether these effects are influenced by human APOE genotype. Transgenic mice expressing human APOE-e3 or e4, (on an APOE-KO background) and a control group of APOE-KO mice underwent transient global ischaemia and two weeks later MHP36 cells were transplanted unilaterally into the striatum and hippocampus. 1 week after grafting the mice were started on a series of tests for motor balance and coordination using the rotarod, and taken for histology 4 weeks post-transplantation. MHP36 graft survival was significantly improved in APOE-e3 mice compared to APOE-KO and APOE-e4 mice. However, the migration and differentiation of MHP36 cells and motor performance of grafted mice were similar in all three APOE groups, indicating a comparable fate and functional activity within a 4 week survival time. Thus the data indicate that APOE genotype may influence cell survival with minimal effect on stem cell migration and differentiation. The data presented in this thesis demonstrate that endogenous apoE strongly influences MHP36 graft survival, migration and differentiation. Although there was minimal evidence that human APOE genotype influences cell migration and differentiation, stem cell survival was markedly improved in a human APOE-e3 allelic environment, which may affect the effectiveness of stem cells in APOE-e4 individuals

FGF19 Regulates Cell Proliferation, Glucose and Bile Acid Metabolism via FGFR4-Dependent and Independent Pathways

Fibroblast growth factor 19 (FGF19) is a hormone-like protein that regulates carbohydrate, lipid and bile acid metabolism. At supra-physiological doses, FGF19 also increases hepatocyte proliferation and induces hepatocellular carcinogenesis in mice. Much of FGF19 activity is attributed to the activation of the liver enriched FGF Receptor 4 (FGFR4), although FGF19 can activate other FGFRs in vitro in the presence of the coreceptor βKlotho (KLB). In this report, we investigate the role of FGFR4 in mediating FGF19 activity by using Fgfr4 deficient mice as well as a variant of FGF19 protein (FGF19v) which is specifically impaired in activating FGFR4. Our results demonstrate that FGFR4 activation mediates the induction of hepatocyte proliferation and the suppression of bile acid biosynthesis by FGF19, but is not essential for FGF19 to improve glucose and lipid metabolism in high fat diet fed mice as well as in leptin-deficient ob/ob mice. Thus, FGF19 acts through multiple receptor pathways to elicit pleiotropic effects in regulating nutrient metabolism and cell proliferation

Driven interfaces in disordered media: determination of universality classes from experimental data

While there have been important theoretical advances in understanding the universality classes of interfaces moving in porous media, the developed tools cannot be directly applied to experiments. Here we introduce a method that can identify the universality class from snapshots of the interface profile. We test the method on discrete models whose universality class is well known, and use it to identify the universality class of interfaces obtained in experiments on fluid flow in porous media.Comment: 4 pages, 5 figure

Multi-scale modeling study of the source contributions to near-surface ozone and sulfur oxides levels over California during the ARCTAS-CARB period

Chronic high surface ozone (O3) levels and the increasing sulfur oxides (SOx = SO2+SO4) ambient concentrations over South Coast (SC) and other areas of California (CA) are affected by both local emissions and long-range transport. In this paper, multi-scale tracer, full-chemistry and adjoint simulations using the STEM atmospheric chemistry model are conducted to assess the contribution of local emission sourcesto SC O3 and to evaluate the impacts of transported sulfur and local emissions on the SC sulfur budgetduring the ARCTAS-CARB experiment period in 2008. Sensitivity simulations quantify contributions of biogenic and fire emissions to SC O3 levels. California biogenic and fire emissions contribute 3–4 ppb to near-surface O3 over SC, with larger contributions to other regions in CA. During a long-range transport event from Asia starting from 22 June, high SOx levels (up to ~0.7 ppb of SO2 and ~1.3 ppb of SO4) is observed above ~6 km, but they did not affect CA surface air quality. The elevated SOx observed at 1–4 km is estimated to enhance surface SOx over SC by ~0.25 ppb (upper limit) on ~24 June. The near-surface SOx levels over SC during the flight week are attributed mostly to local emissions. Two anthropogenic SOx emission inventories (EIs) from the California Air Resources Board (CARB) and the US Environmental Protection Agency (EPA) are compared and applied in 60 km and 12 km chemical transport simulations, and the results are compared withobservations. The CARB EI shows improvements over the National Emission Inventory (NEI) by EPA, but generally underestimates surface SC SOx by about a factor of two. Adjoint sensitivity analysis indicated that SO2 levels at 00:00 UTC (17:00 local time) at six SC surface sites were influenced by previous day maritime emissions over the ocean, the terrestrial emissions over nearby urban areas, and by transported SO2 from the north through both terrestrial and maritime areas. Overall maritime emissions contribute 10–70% of SO2 and 20–60% fine SO4 on-shore and over the most terrestrial areas, with contributions decreasing with in-land distance from the coast. Maritime emissions also modify the photochemical environment, shifting O3 production over coastal SC to more VOC-limited conditions. These suggest an important role for shipping emission controls in reducing fine particle and O3concentrations in SC

Moringa oleifera Improves Skeletal Muscle Metabolism and Running Performance in Mice

Background: Recent estimates suggest that 7% of Americans use plant-derived nutritional supplements to treat a variety of complications and/or to improve athletic performance and skeletal muscle health. Unfortunately, these supplements are largely unregulated and understudied. For example, Moringa oleifera (M. oleifera) is a subtropical plant and is routinely used to treat inflammation, diabetes, obesity, cancer and HIV. However, the mechanism of action of M. oleifera has not been fully elucidated, thus the purpose of this study is to evaluate the role of M. oleifera as a novel ergogenic aid to improve exercise performance by driving peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)-dependent signaling pathways implicated in mitochondrial biogenesis and oxidative metabolism in skeletal muscle tissue. Methods: Adult male C57BL/6 mice were treated with 1.0 g of M. oleifera (N = 20) per day or vehicle control (N = 20) for a total of 5 weeks. Following 3 weeks of supplementation, half of each group (RUN) was given access to running wheels every night for 2 weeks (Remaining half = SED), distances ran were recorded daily. After treatment protocols were complete, the gastrocnemius muscles were excised and assayed for known markers of mitochondrial biogenesis, angiogenesis, endurance capacity, and capillary density using immunohistochemistry and RT-PCR. Results: Our results showed a significant increase in average distance run in the M. oleifera + SED and M. oleifera + RUN groups. This physiological trend was consistent with the molecular profile of key metabolic markers, i.e., there was an increase in levels of PGC-1α, PPARγ, SDHB, SUCLG1, VEGF, PGAM-2, PGK1, and MYLPF in the M. oleifera treated groups compared to vehicle + SED. Moreover, M. oleifera also increased CSA and decreased markers of protein degradation. Conclusions: This data suggests that M. oleifera has the potential to be an ergogenic aid via enhancing energy metabolism in adult skeletal muscle by increasing the expression of key metabolic markers, including those involved in glycolysis, oxidative phosphorylation, mitochondrial biogenesis and angiogenesis

The SCottish Alcoholic Liver disease Evaluation: a population-level matched cohort study of hospital-based costs, 1991-2011

Studies assessing the costs of alcoholic liver disease are lacking. We aimed to calculate the costs of hospitalisations before and after diagnosis compared to population controls matched by age, sex and socio-economic deprivation. We aimed to use population level data to identify a cohort of individuals hospitalised for the first time with alcoholic liver disease in Scotland between 1991 and 2011.Incident cases were classified by disease severity, sex, age group, socio-economic deprivation and year of index admission. 5 matched controls for every incident case were identified from the Scottish population level primary care database. Hospital costs were calculated for both cases and controls using length of stay from morbidity records and hospital-specific daily rates by specialty. Remaining lifetime costs were estimated using parametric survival models and predicted annual costs. 35,208 incident alcoholic liver disease hospitalisations were identified. Mean annual hospital costs for cases were 2.3 times that of controls pre diagnosis (£804 higher) and 10.2 times (£12,774 higher) post diagnosis. Mean incident admission cost was £6,663. Remaining lifetime cost for a male, 50-59 years old, living in the most deprived area diagnosed with acoholic liver disease was estimated to be £65,999 higher than the matched controls (£12,474 for 7.43 years remaining life compared to £1,224 for 21.8 years). In Scotland, alcoholic liver disease diagnosis is associated with significant increases in admissions to hospital both before and after diagnosis. Our results provide robust population level estimates of costs of alcoholic liver disease for the purposes of health-care delivery, planning and future cost-effectiveness analyses

Regulation of Thalamic and Cortical Network Synchrony by Scn8a.

Voltage-gated sodium channel (VGSC) mutations cause severe epilepsies marked by intermittent, pathological hypersynchronous brain states. Here we present two mechanisms that help to explain how mutations in one VGSC gene, Scn8a, contribute to two distinct seizure phenotypes: (1) hypoexcitation of cortical circuits leading to convulsive seizure resistance, and (2) hyperexcitation of thalamocortical circuits leading to non-convulsive absence epilepsy. We found that loss of Scn8a leads to altered RT cell intrinsic excitability and a failure in recurrent RT synaptic inhibition. We propose that these deficits cooperate to enhance thalamocortical network synchrony and generate pathological oscillations. To our knowledge, this finding is the first clear demonstration of a pathological state tied to disruption of the RT-RT synapse. Our observation that loss of a single gene in the thalamus of an adult wild-type animal is sufficient to cause spike-wave discharges is striking and represents an example of absence epilepsy of thalamic origin

Dominance of multidrug resistant CC271 clones in macrolide-resistant streptococcus pneumoniae in Arizona

<p>Abstract</p> <p>Background</p> <p>Rates of resistance to macrolide antibiotics in <it>Streptococcus pneumoniae </it>are rising around the world due to the spread of mobile genetic elements harboring <it>mef</it>(E) and <it>erm</it>(B) genes and post-vaccine clonal expansion of strains that carry them.</p> <p>Results</p> <p>Characterization of 592 clinical isolates collected in Arizona over a 10 year period shows 23.6% are macrolide resistant. The largest portion of the macrolide-resistant population, 52%, is dual <it>mef</it>(E)/<it>erm</it>(B)-positive. All dual-positive isolates are multidrug-resistant clonal lineages of Taiwan^19F-14, mostly multilocus sequence type 320, carrying the recently described transposon Tn<it>2010</it>. The remainder of the macrolide resistant <it>S. pneumoniae </it>collection includes 31% <it>mef</it>(E)-positive, and 9% <it>erm</it>(B)-positive strains.</p> <p>Conclusions</p> <p>The dual-positive, multidrug-resistant <it>S. pneumoniae </it>clones have likely expanded by switching to non-vaccine serotypes after the heptavalent pneumococcal conjugate vaccine release, and their success limits therapy options. This upsurge could have a considerable clinical impact in Arizona.</p