The histone tails have for decades been regarded as unstructured polypeptide chains which simply served as molecular beacons to protein effectors which modify chromatin. However some experimental evidence shows that the tails may contain structure. Thus we conducted a Molecular Dynamics study of the Histone H3 tail and itâs most important post translationally modified isoforms. The 500 ns experiments showed the evolution of different secondary structure conformations for the different modified isoforms. More interestingly the active isoform showed a statistically significant longer reach compared to the inactive isoform. We next conducted a molecular docking study of the 15 â residue tip of the H3 tail to the nucleosome surface. The starting structures were sampled from the Molecular Dynamics trajectories. The tips showed binding to nucleosome where the H3 tail exits the nucleosome, between the DNA and the octamer. This binding position did not cha nge between the different isoforms. We thus propose a molecular mechanism whereby chromatin compaction is carried out at a nucleosome level, and is regulated by transitions in the N-terminal H3 tail structures, which, in turn, are modulated by specific epigenetic PTM patterns.

The mining industry has provided researchers access to the deep subsurface. The deep subsurface is known to harbor a treasure of novel genes and proteins that can be exploited in the biotechnology industry. It has been established that microorganisms in the deep subsurface are potentially novel and are able to endure high temperatures and extreme pH with limited nutrients for survival. Unfortunately most of these organisms are unculturable. Due to the lack of nutrients these microorganisms utilize reduced metals and minerals from the environment as a source of survival in a process known as biogeochemical cycling. Two fissure water samples were collected from two borehole sites at the Northam platinum mine and analyzed through molecular approaches. Microbial biodiversity was determined for borehole NO24FW030908 fissure water sample. The microbial biodiversity was based on the 16S rRNA and 18S rRNA gene clone libraries determined through phylogenetic clustering analyses using ARB software and a comparative analysis was done using DGGE profiling. The prokaryote and eukaryote diversity revealed low diversity at the species level but a high intraspecies diversity probably associated with the novelty of the biome. Unique isolates were cultured from borehole NO212FW050508 fissure water sample. Five isolates showed novelty at the species level and one isolate showed novelty at the genus level. Two isolates, Geobacillus sp. A8 and Geobacillus sp. A12 were sent for characterization at the DSMZ, Germany. Both isolates exhibited similarities at the genus level but significant differences at the species level based on the type strain to warrant different taxonomical positions. These isolates along with Thermus scotoductus SA-01 were analyzed for platinum reduction and the possible formation of metallic platinum. All isolates showed the ability to reduce platinum (IV) to platinum (0). Geobacillus sp. A8 was selected for further characterizations of platinum nanoparticle formation. Platinum nanoparticles were characterized with various tools to show the size and shape using TEM and SEM, to show the composition using XRD and EDS, and to show the particle size distribution using the NanoTrac and NiComp 380 ZLS systems. It has been proposed that a classical hydrogenase activated by a cytochrome c3 is responsible for the two-step reduction of platinum. Therefore, hydrogenase inhibition tests and the TTC test for the presence of an active hydrogenase were done to confirm the presence of a hydrogenase in Geobacillus sp. A8. It was then selected for the construction of the metabolic pathway genome database to study the metabolism of the microorganism in order to identify alternate or novel pathway(s) associated with the genome. Platinum reduction activity tests based on subcellular fractionation revealed platinum reduction and deposition that occurred in the periplasm; therefore, the putative protein involved in platinum reduction was probably periplasmic. The periplasmic fraction was separated into three fraction sizes, greater than 30 kDa, between 10 and 30 kDa and less that 10 kDa. The 10 â 30 kDa fraction revealed positive platinum reduction. The active fraction was analyzed on a SDS-PAGE and revealed three bands that were digested by trypsin and the peptides were analyzed by protein mass spectrometry. Two proteins were identified, an oxidoreductase commonly known as the old yellow enzyme previously shown to reduce chromate (VI), and a hypothetical YajQ protein. Both proteins were expressed and purified and both proteins showed the ability to reduce platinum. Further work would be to elucidate the in situ mechanism involved in the reduction of platinum with hydrogen as the electron donor.

The consumption of meat is increasingly linked to various diseases and this has already affected the growth of this sector of the food industry in some countries. Pork is seen as one of the major contributors to this problem. The meat industry reacted by using strategies such as dietary supplementation and direct addition of healthier lipids to manipulate the nutritional value of meat. The positive effects of CLA on human health are well documented and various strategies have been successfully employed in increasing the levels of CLA in different animal models such as pigs and eventually pork products. The effects CLA may have on a fermented meat product like salami has not been studied yet. No research have been reported where it was attempted to increase the nutritional value of salami, maintain acceptable product quality and include a therapeutically high level of CLA with the belief that it will benefit human health. In the first experiment of this study, 40 Duroc X Landrace gilts weighing on average 35 kg were randomly divided into two groups fed either a diet containing 0.5% sunflower oil (SFO) or a diet containing 0.5% conjugated linoleic acid (Luta-CLA® 60, BASF). These groups were further divided into two slaughter weight groups of ±70 kg and ±90 kg. After slaughter the lean meat and backfat from the loins of these animals were pooled by treatment group and utilized to manufacture salami. The aim was to determine if salami quality is influenced by slaughter weight and dietary supplementation of CLA. Both variables had major effects on the fatty acid composition and fatty acid ratios of the muscle and fat raw material as well as salami. The fatty acids and fatty acid ratios of technological importance were mostly positively influenced while the fatty acids and fatty acid ratios of nutritional and health concern were mostly negatively influenced by increased slaughter weight and dietary CLA supplementation. The microbial, physical, sensory and lipid stability parameters of salami were unaffected or inconsistently affected by both variables. Although dietary CLA was deposited successfully in muscle and fat, the deposition level was low. Consumption of a 28 g portion of salami manufactured from CLA enriched pork could only supply in 1% of the RDA for CLA. It could be concluded that although dietary supplementation of pork with CLA improved the technological properties of fat tissue it could not be considered a very successful approach to increase human consumption of CLA. In the second experiment of this study the aim was to increase the CLA content of salami to three different percentages (25%, 50% and 100%) of the RDA for CLA per 28 g portion of salami. This was accomplished through the direct addition of CLA (Tonalin® TG 80) in a pre-emulsified form with proportional decreases in the normally used pork BF content of the salamis. The salamis from these three treatment groups were then compared to a 100% pork BF control group for any possible effects on the microbial, physical and lipid stability parameters as well as fatty acid composition and fatty acid ratios. Microbial and sensory parameters were largely unaffected with varying effects on the physical and lipid stability parameters. Major effects on the fatty acid composition and fatty acid ratios of the salamis were observed. The partial replacement of pork BF and direct addition of CLA to salami proved to be an effective method of increasing CLA levels in salami in an attempt to improve the health aspects of salami to the point where it could be regarded as a functional food.

No description available.

Luminescent transition metal complexes have arisen as viable alternatives to organic dyes for sensory applications due to their notable advantages. This thesis aimed to synthesize different kinds of iridium(III) complexes, explore their interactions with DNAs and investigate their application for the construction of oligonucleotide-based sensing platforms for important biomarkers. A series of iridium(III) complexes incorporating a variety of C^N and N^N donor ligands were synthesized and were demonstrated to possess G-quadruplex-selective binding properties via emission titration, UV/vis titration, fluorescence resonance energy transfer melting and G-quadruplex fluorescent intercalator displacement experiments. These G-quadruplex-selective iridium(III) complexes were utilized as signal transducers to monitor the conformational changes of oligonucleotides in oligonucleotide-based luminescent detection platforms for protein tyrosine kinase-7, interferon-gamma, sialic acidbinding immunoglobulin-likelectin-5 and thymine DNA glycosylase. And these designed platforms could work effectively in the diluted cell extract as the results in this thesis indicated.

Autoimmune diseases and mood-related disorders are among the major plagues of our modern society, as they impair the normal daily life of patients at both social and physical levels. Autoimmunity is caused by the loss of immunological tolerance i.e. the inability of immune cells to make a distinction between self and non-self antigens. Intriguingly, patients suffering from autoimmune diseases also show higher rate of “unjustified” mood disorders, such as depression and anxiety. Recent evidence indicates mood disorders as biomarkers for autoimmunity rather than co-morbidities since their occurrence is unrelated to the time of diagnosis of the autoimmune disease or to the degree of disability. In this thesis I investigated the phenotype of T cell-specific transgenic mice overexpressing Annexin-A1 (AnxA1tg): homeostatic immunomodulatory protein with dual opposite functions in the innate and adaptive immune system. Consistent with the previously observed pro-inflammatory role in T cells, AnxA1tg mice showed higher susceptibility to develop autoimmune diseases like multiple sclerosis and systemic lupus erythematosus. Most interestingly, using a battery of behavioural tests, we showed an increased anxious-like behaviour in AnxA1tg mice compared to wild type. This phenotype was associated with a specific gene pattern in the brain and in T cells as shown by microarray analyses. Adoptive transfer of AnxA1tg-CD4+ T cells into wild-type mice caused an increased anxiety-like behaviour in the recipient animals thus providing first experimental evidence for emotional dysfunction in autoimmunity-prone animals. In conclusion, the results of this study provide novel evidences for the strong link between immune system and CNS. More specifically, our findings highlight a novel function of CD4+ T cells as the drivers of mental and physical wellbeing. Future studies will assess the potential of strategies targeting AnxA1 in T cells as new therapeutic tools for the combined treatment for autoimmunity and associated mental disorders.

A school of literature has shown that Annexin-A1 (Anx-A1) is an endogenous anti-inflammatory protein that exerts a regulatory control over the innate immune system in order to restore homeostasis after an inflammatory reaction. Surprisingly, recent published works have highlighted that Anx-A1 has an alternate role in the adaptive immune system by positively modulating the strength of TCR signalling and biasing helper-subset differentiation. Dendritic cells are a class of innate leukocytes, poised at the environmental interface, that are the essential immune cells responsible in the initiation of T-cell driven responses. These findings provided the foundation for this PhD project, the principal aim of which is to provide a link between the disparate effects of Annexin-A1 in innate and adaptive immunity by investigating the role of endogenous Annexin-A1 in dendritic cell biology and its effector function as an antigen-presenting cell towards T cell activation and differentiation. To address this hypothesis, I cultured bone marrowderived dendritic cells from AnxA1-deficient mice or control littermates and stimulated with LPS (100ng/ml) then compared phenotypic and functional characteristics. My results demonstrate that Anx-A1-/- bone marrow derived dendritic cells show an increased number of CD11c+ cells expressing high levels of some maturation markers such as CD40, CD54 and CD80 and a decreased capacity to take up antigen compared to control Anx-A1+/+ cells. However, analysis of LPS-treated dendritic cells from Anx-A1-/- mice demonstrated a diminished up-regulation of maturation markers, a decreased migratory activity in vivo and an attenuated production of the inflammatory cytokines Interleukin (IL)- 1β, Tumour Necrosis Factor (TNF)-α and IL-12. This defect was resultant of an impaired Nuclear Factor (NF)-κB/DNAbinding activity due to lack of Anx-A1 signalling as demonstrated by the reduced activation of Extracellular-signal Regulated Kinase (ERK) 1/2 and protein kinase B (PKB)/Akt compared to cells from control littermates. As a consequence of these defects, I assessed the antigenpresenting/ T-cell activating capabilities of these DC. Anx-A1-/- DC showed an impaired capacity to stimulate T cell proliferation and differentiation in allogeneic mixed leukocyte reaction. To dissect this biologically relevant phenomenon further, I employed an antigenspecific, T-cell restricted model; a co-culture system of chicken ovalbumin peptide-pulsed, LPS-matured bone marrow-derived DC incubated with transgenic TCR T cells from OT-I/RAG-1-/- (OT-I, OTI/ CD8+) or OT-II/ RAG-1-/- (OT-II, OT-II/CD4+) mice. Peptide-pulsed, LPS-matured AnxA1-/- DC failed to initiate an appropriate T cell activation in both OT-I and OT-II T cells indicated by reduced cell proliferation when compared to T cells co-cultured with peptide6 pulsed, LPS-matured AnxA1+/+ DC. Additionally, comparison of peptide-pulsed, LPS-matured AnxA1-/- DC with AnxA1+/+ DC counterparts detected severely diminished levels of IL-2 from cocultures with OT-I T cells and ablated IFN-γ production from cocultures with both OT-I and OT-II T cells. In conclusion, AnxA1 seems to act as a positive modulator of immunogenic activation of DC, whereby the AnxA1 signal pathway has a probable synergism with the TLR4 signalling cascade. DCderived AxnA1 appears to contribute in promoting T cell activation with a larger influence on OT-I/CD8+ T cells than OT-II/CD4+ T cells. Altogether these findings suggest that inhibition of Anx-A1 expression or function in dendritic cells might represent a useful way to modulate the adaptive immune response and pathogen-induced T cell-driven immune diseases.

Microparticles (MP) are submicron structures produced by all cells upon activation or apoptosis that act as a non-soluble means of communication between cells. They ferry proteins, bioactive lipids, RNA and receptors, as well as ridding cells of redundant organelles and toxins. They have been recently investigated for their pathophysiological role and as potential biomarkers/effectors in many diseases. In severe sepsis, studies of MP so far have produced inconsistent and even conflicting results. In this project, it was demonstrated that cell derived MP subsets vary according to the cause of severe sepsis (community acquired pneumonia (CAP) or faecal peritonitis (FP)), where CAP patients had higher levels of circulating MP. Surprisingly, FP patients MP levels were comparable to healthy volunteers. Further stratification of MP subsets according to their expression of the protein alpha-2-macroglobulin (A2M) has yielded better differentiation between the two diseases. The A2M expressing MP were significantly higher in survivors of community acquired pneumonia sepsis, but there was no similar association in patient with FP. Granulocyte macrophage colony-stimulating factor (GM-CSF) and interferon γ (IFN-γ) are being studied as possible adjuvant therapies in sepsis. They seem to reverse the immune-paresis that ensues after the initial insult. MP produced from whole blood stimulated with GM-CSF and IFN-γ were studied in this project. Both GM-CSF and IFN-γ increased MP expressing A2M over control. These MP elicited a pro-inflammatory phenotype when incubated with neutrophils or endothelial cells which may contribute to the potential benefits of GM-CSF and IFN-γ in severe sepsis.

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Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 228-239). Also available in electronic version.