Immunomodulatory effects exerted by Lactobacillus salivarius strains on innate immune cells

Despite increased application of commensal bacteria for attempting to improve the symptoms of a variety of inflammatory conditions, including inflammatory bowel diseases, diarrhoea and irritable bowel syndrome, therapeutic approaches that involve live bacteria are hampered by a limited understanding of bacterium-host interactions. Lactobacilli are natural inhabitants of the mammalian gastrointestinal tract and many lactobacilli are regarded as probiotics meaning that they exert a beneficial influence on the health status of their consumers. Modulation of immune responses is a plausible mechanism underlying these beneficial effects. The aim of this thesis was to investigate the effect of 33 Lactobacillus salivarius strains on the production of inflammatory cytokines from a variety of human and mouse immune cells. Induction of immune responses in vitro was shown to be bacterial- and mouse strain-dependent, cell type-dependent, blood donor-dependent and bacterial cell number-dependent. Collectively, these data suggest the importance of a case-by-case selection of candidate strains for their potential therapeutic application. Toll-like receptors (TLRs) recognize microbe-associated molecular patterns (MAMPs) and play a critical role in shaping microbial-specific innate and adaptive immune responses. Following ligand engagement, TLRs trigger a complex network of signalling that culminate in the production of inflammatory mediators. The investigation of the molecular mechanisms underlying the Lb. salivarius-host interaction resulted in the identification of a novel role for TLR2 in negatively regulating TLR4 signalling originated from subcellular compartments within macrophages. Notably, sustained activation of JAK/STAT cascade and M1-signature genes in TLR2-/- macrophages was ablated by selective TLR4 and JAK inhibitors and by absence of TLR4 in TLR2/4-/- cells. In addition, other negative regulators of TLR signalling triggered by Lb. salivarius strains were found to be the adapter molecules TIRAP and TRIF. Understanding negative regulation of TLR signalling may pave the way for the development of novel therapeutics to limit inflammation in multiple diseases.

Investigations into the cellular and molecular changes in the amygdala in models of temporal lobe epilepsy and maternal immune activation

The amygdala is a limbic structure that is involved in many of our emotions and processing of these emotions such as fear, anger and pleasure. Conditions such as anxiety, autism, and also epilepsy, have been linked to abnormal functioning of the amygdala, owing to improper neurodevelopment or damage. This thesis investigated the cellular and molecular changes in the amygdala in models of temporal lobe epilepsy (TLE) and maternal immune activation (MIA). The kainic acid (KA) model of temporal lobe epilepsy (TLE) was used to induce Ammon’s-horn sclerosis (AHS) and to investigate behavioural and cytoarchitectural changes that occur in the amygdala related to Neuropeptide Y1 receptor expression. Results showed that KA-injected animals showed increased anxiety-like behaviours and displayed histopathological hallmarks of AHS including CA1 ablation, granule cell dispersion, volume reduction and astrogliosis. Amygdalar volume and neuronal loss was observed in the ipsilateral nuclei which was accompanied by astrogliosis. In addition, a decrease in Y1 receptor expressing cells in the ipsilateral CA1 and CA3 sectors of the hippocampus, ipsi- and contralateral granule cell layer of the dentate gyrus and ipsilateral central nucleus of the amygdala was found, consistent with a reduction in Y1 receptor protein levels. The results suggest that plastic changes in hippocampal and/or amygdalar Y1 receptor expression may negatively impact anxiety levels. Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain and tight regulation and appropriate control of GABA is vital for neurochemical homeostasis. GABA transporter-1 (GAT-1) is abundantly expressed by neurones and astrocytes and plays a key role in GABA reuptake and regulation. Imbalance in GABA homeostasis has been implicated in epilepsy with GAT-1 being an attractive pharmacological target. Electron microscopy was used to examine the distribution, expression and morphology of GAT-1 expressing structures in the amygdala of the TLE model. Results suggest that GAT-1 was preferentially expressed on putative axon terminals over astrocytic processes in this TLE model. Myelin integrity was examined and results suggested that in the TLE model myelinated fibres were damaged in comparison to controls. Synaptic morphology was studied and results suggested that asymmetric (excitatory) synapses occurred more frequently than symmetric (inhibitory) synapses in the TLE model in comparison to controls. This study illustrated that the amygdala undergoes ultrastructural alterations in this TLE model. Maternal immune activation (MIA) is a risk factor for neurodevelopmental disorders such as autism, schizophrenia and also epilepsy. MIA was induced at a critical window of amygdalar development at E12 using bacterial mimetic lipopolysaccharide (LPS). Results showed that MIA activates cytokine, toll-like receptor and chemokine expression in the fetal brain that is prolonged in the postnatal amygdala. Inflammation elicited by MIA may prime the fetal brain for alterations seen in the glial environment and this in turn have deleterious effects on neuronal populations as seen in the amygdala at P14. These findings may suggest that MIA induced during amygdalar development may predispose offspring to amygdalar related disorders such as heightened anxiety, fear impairment and also neurodevelopmental disorders.

Identifying novel molecular mechanisms of ghrelin receptor signalling underlying neural control of food intake: interaction with stress and impulsivity

The gut-hormone, ghrelin, activates the centrally expressed growth hormone secretagogue 1a (GHS-R1a) receptor, or ghrelin receptor. The ghrelin receptor is a G-protein coupled receptor (GPCR) expressed in several brain regions, including the arcuate nucleus (Arc), lateral hypothalamus (LH), ventral tegmental area (VTA), nucleus accumbens (NAcc) and amygdala. Activation of the GHS-R1a mediates a multitude of biological activities, including release of growth hormone and food intake. The ghrelin signalling system also plays a key role in the hedonic aspects of food intake and activates the dopaminergic mesolimbic circuit involved in reward signalling. Recently, ghrelin has been shown to be involved in mediating a stress response and to mediate stress-induced food reward behaviour via its interaction with the HPA-axis at the level of the anterior pituitary. Here, we focus on the role of the GHS-R1a receptor in reward behaviour, including the motivation to eat, its anxiogenic effects, and its role in impulsive behaviour. We investigate the functional selectivity and pharmacology of GHS-R1a receptor ligands as well as crosstalk of the GHS-R1a receptor with the serotonin 2C (5-HT2C) receptor, which represent another major target in the regulation of eating behaviour, stress-sensitivity and impulse control disorders. We demonstrate, to our knowledge for the first time, the direct impact of GHS-R1a signalling on impulsive responding in a 2-choice serial reaction time task (2CSRTT) and show a role for the 5-HT2C receptor in modulating amphetamine-associated impulsive action. Finally, we investigate differential gene expression patterns in the mesocorticolimbic pathway, specifically in the NAcc and PFC, between innate low- and high-impulsive rats. Together, these findings are poised to have important implications in the development of novel treatment strategies to combat eating disorders, including obesity and binge eating disorders as well as impulse control disorders, including, substance abuse and addiction, attention deficit hyperactivity disorder (ADHD) and mood disorders.

Multiple Dynamic Processes Contribute to the Complex Steady Shear Behavior of Cross-Linked Supramolecular Networks of Semidilute Entangled Polymer Solutions.

Molecular theories of shear thickening and shear thinning in associative polymer networks are typically united in that they involve a single kinetic parameter that describes the network -- a relaxation time that is related to the lifetime of the associative bonds. Here we report the steady-shear behavior of two structurally identical metallo-supramolecular polymer networks, for which single-relaxation parameter models break down in dramatic fashion. The networks are formed by the addition of reversible cross-linkers to semidilute entangled solutions of PVP in DMSO, and they differ only in the lifetime of the reversible cross-links. Shear thickening is observed for cross-linkers that have a slower dissociation rate (17 s(-1)), while shear thinning is observed for samples that have a faster dissociation rate (ca. 1400 s(-1)). The difference in the steady shear behavior of the unentangled vs. entangled regime reveals an unexpected, additional competing relaxation, ascribed to topological disentanglement in the semidilute entangled regime that contributes to the rheological properties.

Where does energy R&D come from? Examining crowding out from energy R&D

Recent efforts to endogenize technological change in climate policy models demonstrate the importance of accounting for the opportunity cost of climate R&D investments. Because the social returns to R&D investments are typically higher than the social returns to other types of investment, any new climate mitigation R&D that comes at the expense of other R&D investment may dampen the overall gains from induced technological change. Unfortunately, there has been little empirical work to guide modelers as to the potential magnitude of such crowding out effects. This paper considers both the private and social opportunity costs of climate R&D. Addressing private costs, we ask whether an increase in climate R&D represents new R&D spending, or whether some (or all) of the additional climate R&D comes at the expense of other R&D. Addressing social costs, we use patent citations to compare the social value of alternative energy research to other types of R&D that may be crowded out. Beginning at the industry level, we find no evidence of crowding out across sectors-that is, increases in energy R&D do not draw R&D resources away from sectors that do not perform R&D. Given this, we proceed with a detailed look at alternative energy R&D. Linking patent data and financial data by firm, we ask whether an increase in alternative energy patents leads to a decrease in other types of patenting activity. While we find that increases in alternative energy patents do result in fewer patents of other types, the evidence suggests that this is due to profit-maximizing changes in research effort, rather than financial constraints that limit the total amount of R&D possible. Finally, we use patent citation data to compare the social value of alternative energy patents to other patents by these firms. Alternative energy patents are cited more frequently, and by a wider range of other technologies, than other patents by these firms, suggesting that their social value is higher. © 2011 Elsevier B.V.

Characterization of topographical effects on macrophage behavior in a foreign body response model.

Current strategies to limit macrophage adhesion, fusion and fibrous capsule formation in the foreign body response have focused on modulating material surface properties. We hypothesize that topography close to biological scale, in the micron and nanometric range, provides a passive approach without bioactive agents to modulate macrophage behavior. In our study, topography-induced changes in macrophage behavior was examined using parallel gratings (250 nm-2 mum line width) imprinted on poly(epsilon-caprolactone) (PCL), poly(lactic acid) (PLA) and poly(dimethyl siloxane) (PDMS). RAW 264.7 cell adhesion and elongation occurred maximally on 500 nm gratings compared to planar controls over 48 h. TNF-alpha and VEGF secretion levels by RAW 264.7 cells showed greatest sensitivity to topographical effects, with reduced levels observed on larger grating sizes at 48 h. In vivo studies at 21 days showed reduced macrophage adhesion density and degree of high cell fusion on 2 mum gratings compared to planar controls. It was concluded that topography affects macrophage behavior in the foreign body response on all polymer surfaces examined. Topography-induced changes, independent of surface chemistry, did not reveal distinctive patterns but do affect cell morphology and cytokine secretion in vitro, and cell adhesion in vivo particularly on larger size topography compared to planar controls.

Waveguide QED: Many-body bound-state effects in coherent and Fock-state scattering from a two-level system

Strong coupling between a two-level system (TLS) and bosonic modes produces dramatic quantum optics effects. We consider a one-dimensional continuum of bosons coupled to a single localized TLS, a system which may be realized in a variety of plasmonic, photonic, or electronic contexts. We present the exact many-body scattering eigenstate obtained by imposing open boundary conditions. Multiphoton bound states appear in the scattering of two or more photons due to the coupling between the photons and the TLS. Such bound states are shown to have a large effect on scattering of both Fock- and coherent-state wave packets, especially in the intermediate coupling-strength regime. We compare the statistics of the transmitted light with a coherent state having the same mean photon number: as the interaction strength increases, the one-photon probability is suppressed rapidly, and the two- and three-photon probabilities are greatly enhanced due to the many-body bound states. This results in non-Poissonian light. © 2010 The American Physical Society.

Surface physics at the nano-scale via scanning probe microscopy and molecular dynamics simulations

Probe-based scanning microscopes, such as the STM and the AFM, are used to obtain the topographical and electronic structure maps of material surfaces, and to modify their morphologies on nanoscopic scales. They have generated new areas of research in condensed matter physics and materials science. We will review some examples from the fields of experimental nano-mechanics, nano-electronics and nano-magnetism. These now form the basis of the emerging field of Nano-technology. A parallel development has been brought about in the field of Computational Nano-science, using quantum-mechanical techniques and computer-based numerical modelling, such as the Molecular Dynamics (MD) simulation method. We will report on the simulation of nucleation and growth of nano-phase films on supporting substrates. Furthermore, a theoretical modelling of the formation of STM images of metallic clusters on metallic substrates will also be discussed within the non-equilibrium Keldysh Green function method to study the effects of coherent tunnelling through different atomic orbitals in a tip-sample geometry.

Effects of reflow process on the reliability of flip chip on flex interconnections using anisotropic conductive adhesives

In this paper, the effects of the solder reflow process on the reliability of anisotropic conductive film (ACF) interconnections for flip chip on flex (FCOF) applications are investigated. Experiments as well as computer modeling methods have been used. In the experiments, it was found that the contact resistance of ACF joints increased after the subsequent reflow process, and the magnitude of this increase was strongly correlated to the peak temperature of the reflow profile. Nearly 40% of the joints were opened (i.e. lifted away from the pad) after the reflow process with 260 °C peak temperature while no opening was observed when the peak temperature was 210 °C. It is believed that the CTE mismatch between the polymer particle and the adhesive matrix is the main cause of this contact degradation. It was also found that the ACF joints after the reflow process with 210 °C peak temperature showed a high ability to resist water absorption under steady state 85 °C/85%RH conditions, probably because the curing degree of the ACF was improved during the reflow process. To give a good understanding, a 3D model of an ACF joint structure was built and finite element analysis was used to predict the stress distribution in the conductive particles, adhesive matrix and metal pads during the reflow process.

Effects of solder reflow on the reliability of flip-chip on flex interconnections using anisotropic conductive adhesives

This work describes the work of an investigation of the effects of solder reflow process on the reliability of anisotropic conductive film (ACF) interconnection for flip-chip on flex (FCOF) applications. Experiments as well as computer modeling methods have been used. The results show that the contact resistance of ACF interconnections increases after the reflow and the magnitude of the increase is strongly correlated to the peak reflow temperature. In fact, nearly 40 percent of the joints are open when the peak reflow temperature is 260°C, while there is no opening when the peak temperature is 210°C. It is believed that the coefficient of thermal expansion (CTE) mismatch between the polymer particle and the adhesive matrix is the main cause of this contact degradation. To understand this phenomenon better, a three-dimensional (3-D) finite element (FE) model of an ACF joint has been analyzed in order to predict the stress distribution in the conductive particles, adhesive matrix and metal pads during the reflow process. The stress level at the interface between the particle and its surrounding materials is significant and it is the highest at the interface between the particle and the adhesive matrix.

The use of fluorescence microscopy to define polymer localisation to the late endocytic compartments in cells that are targets for drug delivery

Macromolecular therapeutics and nano-sized drug delivery systems often require localisation to specific intracellular compartments. In particular, efficient endosomal escape, retrograde trafficking, or late endocytic/lysosomal activation are often prerequisites for pharmacological activity. The aim of this study was to define a fluorescence microscopy technique able to confirm the localisation of water-soluble polymeric carriers to late endocytic intracellular compartments. Three polymeric carriers of different molecular weight and character were studied: dextrin (Mw~50,000 g/mol), a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (Mw approximately 35,000 g/mol) and polyethylene glycol (PEG) (Mw 5000 g/mol). They were labelled with Oregon Green (OG) (0.3-3 wt.%; <3% free OG in respect of total). A panel of relevant target cells were used: THP-1, ARPE-19, and MCF-7 cells, and primary bovine chondrocytes (currently being used to evaluate novel polymer therapeutics) as well as NRK and Vero cells as reference controls. Specific intracellular compartments were marked using either endocytosed physiological standards, Marine Blue (MB) or Texas-red (TxR)-Wheat germ agglutinin (WGA), TxR-Bovine Serum Albumin (BSA), TxR-dextran, ricin holotoxin, C6-7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labelled ceramide and TxR-shiga toxin B chain, or post-fixation immuno-staining for early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins (LAMP-1, Lgp-120 or CD63) or the Golgi marker GM130. Co-localisation with polymer-OG conjugates confirmed transfer to discreet, late endocytic (including lysosomal) compartments in all cells types. The technique described here is a particularly powerful tool as it circumvents fixation artefacts ensuring the retention of water-soluble polymers within the vesicles they occupy.

Development and validation of a molecular technique for the analysis of archived formalin-preserved phytoplankton samples permits retrospective assessment of Emiliania huxleyi communities

The coccolithophores, particularly the species Emiliania huxleyi (Lohmann) Hay & Mohler, account for the bulk of global calcium carbonate production and as such play a fundamental role in global CO2 cycling and the carbonate chemistry of the oceans. To evaluate the response of this functional group to the effects of climate change, we undertook a feasibility study to determine whether a retrospective approach could be used on archived coccolithophore datasets. We demonstrate for the first time a technique for the extraction of E. huxleyi nucleic acids from archived formalin-fixed samples of the long-term Continuous Plankton Recorder. Molecular analysis of a nine year old formalin-fixed sample reveals the presence of a diverse population of E. huxleyi genotypes within a developing coccolithophore bloom. In addition, E. huxleyi sequences were amplified from a number of formalin-fixed samples, the earliest of which was collected in August 1972. This molecular assay promises the possibility of studying global variations in the distribution and genetic make-up of E. huxleyi communities over extensive periods of time. (c) 2008 Elsevier B.V. All rights reserved.

Long-term effects of ocean warming on the prokaryotic community: evidence from the vibrios

The long-term effects of ocean warming on prokaryotic communities are unknown because of lack of historical data. We overcame this gap by applying a retrospective molecular analysis to the bacterial community on formalin-fixed samples from the historical Continuous Plankton Recorder archive, which is one of the longest and most geographically extensive collections of marine biological samples in the world. We showed that during the last half century, ubiquitous marine bacteria of the Vibrio genus, including Vibrio cholerae, increased in dominance within the plankton-associated bacterial community of the North Sea, where an unprecedented increase in bathing infections related to these bacteria was recently reported. Among environmental variables, increased sea surface temperature explained 45% of the variance in Vibrio data, supporting the view that ocean warming is favouring the spread of vibrios and may be the cause of the globally increasing trend in their associated diseases.

Processing, Annealing and Sterilisation of Poly-L-Lactide.

Poly--lactide (PLLA) is one of the most significant members of a group of polymers regarded as bioabsorbable. Degradation of PLLA proceeds through hydrolysis of the ester bonds in the polymer chains and is influenced significantly by the polymer's molecular weight and crystallinity. To evaluate the effects of processing and sterilisation on these properties, PLLA pellets were either compression moulded or extruded, subjected to annealing at 120°C for 4 h and sterilised by ethylene oxide (EtO) gas. Procedures were used to evaluate the mechanical properties, molecular weight and crystallinity. Upon processing, the crystallinity of the material fell from 61% for the PLLA pellets to 12% and 20% for the compressed and extruded components, respectively. After annealing, crystallinity increased to 43% for the compression-moulded material and 40% for the extruded material. Crystallinity further increased upon EtO sterilisation. A slight decrease in molecular weight was observed for the extruded material through processing, annealing and sterilisation. Young's modulus generally increased with increasing crystallinity, and extension at break and tensile strength decreased. The results from this investigation suggest that PLLA is sensitive to processing and sterilisation, altering properties critical to its degradation rate.