Morphological and Proliferative Responses of Rat Microglia Cells to Lipopolysaccharide and Β-Amyloid

Microglia are the residential macrophages of the central nervous system (CNS) and are sensitive to any changes in their environment. There is rapid microglial activation in most pathological conditions in CNS. The association of activated microglia with various inflammatory and neurodegenerative diseases are well documented. In this study we evaluated the in vitro responses of microglia to lipopolysaccharide (LPS) or beta amyloid (Aβ) in an attempt to further define the activation of microglia. LPS is a potent activator of monocytes and macrophages and Aβ is used to activate microglia in vitro into a neurotoxic phenotype distinguished by secretion of proinflammatory molecules. Microglia were cultured from Sprague-Dawley neonatal rats and purity of culture determined using a common marker for microglia, lectin. Morphological changes following LPS or Aβ treatment were evaluated using fluorescent and confocal microscope. Treated microglia assumed a deramified shape with a condensed cytoplasm, typical of activated microglia. Stimulation of microglia with LPS or Aβ also resulted in significant ultrastructural changes. Transmission electron microscopy revealed an increased number of enlarged, elongated and swollen mitochondria. There were also some changes in the appearance of the endoplasmic reticulum. Untreated microglia displayed mainly smooth endoplasmic reticulum (SER) whereas LPS-treated cells displayed polyribosomes and rough endoplasmic reticulum (RER). It is therefore assumed that LPS-treated microglia have an increased ability in synthesising protein, some of which may be secretory molecules necessary for inflammation. Aβ-treated microglia also displayed more RER compared to control, but lesser compared to LPS. The nucleus in treated microglia appeared enlarged in comparison to untreated cells. The number of cells following treatment revealed that microglia are more viable following LPS treatment compared to Aβ. Immunophenotyping assays demonstrated upregulation of MHC II and CD40 by microglia following treatment. Both MHC II and CD40 are implicated in antigen-presenting and this result indicates that in comparison to LPS, Aβ may have more ability to induce antigen-presenting capabilities in microglia. As shown by viability counts, carboxy fluorescein succinimidyl ester (CFSE) staining also revealed no microglia proliferation following treatment with LPS and Aβ. In conclusion, our study illustrates activation-induced alterations in the morphology, ultrastructure and cell surface phenotype of microglia, which were not accompanied by proliferation of microglia. These changes represent the range of effects that occur in microglia following activation. In an attempt to culture microglia from adult rats for the purpose of determining the effects of aging on microglia responses, modifications were made on the established protocol for neonatal microglia cultures. This includes growing adult cells on poly-L-lysine-coated tissue culture flasks and substituting insulin in cell culture media with insulin-transferrin-selenium (ITS). We were able to successfully support the in vitro growth of adult microglia, which was also enhanced with the addition of the growth factor M-CSF. Key words: microglia, lipopolysacharide, beta amyloid, morphology, proliferation, CD40/MHC I

A reduced data dynamic energy model of the UK houses

This thesis describes the development of a Reduced Data Dynamic Energy Model (RdDEM) for simulating the energy performance of UK houses. The vast quantity of Energy Performance Certificate (EPC) data stored at the national scale provides an unprecedented data source for energy modelling. The majority of domestic energy models developed for the UK houses in recent years, including the Standard Assessment Procedure (SAP) model used for generating EPCs, employ BREDEM (Building Research Establishment Domestic Energy Model) based steady state calculation engines. These models fail to represent the transient behaviours that occur between building envelope and systems with external weather conditions and occupants. Consequently, there is an ongoing debate over the suitability of such models for policy making decisions; which has raised the interest in dynamic energy models to overcome these shortcomings. The RdDEM eliminates the main drawback associated with dynamic energy modelling, namely the large amount of required input data compared to steady-state models, by enhancing a reduced set of data which was originally collected for EPCs. A number of new inferences and methodological enhancements were tested and implemented in the RdDEM using a sample of semi-detached houses. In this way, SAP equivalent input data could be converted automatically for use in dynamic energy modelling software, EnergyPlus. Simulations of indoor air temperatures and space heating energy demand from the RdDEM were compared to those from SAP for 83 semi-detached houses. The comparison was also carried out with more detailed models, on a sub-set of the modelled dwellings. Finally, the predicted energy savings that resulted from energy efficiency improvements of the dwellings were compared and estimated potential for saving energy from the RdDEM was quantified. ii The results show that it is technically feasible to develop dynamic energy models of these houses using equivalent inputs. In the majority of cases, the RdDEM predicted lower indoor air temperatures than SAP, and consequently the energy demands were lower. The RdDEM predicted annual space heating demand to be lower than SAP in 72% of the houses, however the difference was less than 10% in 94% of the houses. The RdDEM predicted slightly higher (< 2%) energy saving potentials compared to SAP when the same set of energy saving measures were implemented in both models. The development of these new methods for automatically creating SAP equivalent inputs from reduced data but for use in a dynamic energy model offers new opportunities for inter-model comparisons as well as a dynamic alternative to the SAP when variations in energy demand and indoor air temperatures are required

FIRST AMENDMENT PROTECTION FOR COMPUTER ASSISTED DESIGN FILES

This thesis examines the possible extent of First Amendment protection for computer assisted design (CAD) files, the pages of code that instruct a 3-D printer on what to print. This thesis draws a comparison between traditional computer code and CAD files, and first asks whether the factors relied upon by courts in determining whether computer code receives First Amendment protection can be applied to CAD files. This thesis also reviews the foundational theories of the First Amendment, and asks whether CAD files fulfill the objectives of the First Amendment under the reasoning of each theory. It concludes by arguing that CAD files should be treated in the same manner as computer code – receiving presumptive protection under the First Amendment, and requiring that any regulation or restriction be content-neutral and narrowly tailored.Master of Art