ROLE OF MULTIPLE TOLL-LIKE RECEPTOR ACTIVATION IN REGULATING CYTOTOXIC T CELL PRIMING TO LYMPOCYTIC CHORIOMENINGITIS VIRUS INFECTIONS

Foreign pathogens are recognized by toll-like receptors (TLR), present on various immune cells such as professional antigen-presenting cells (pAPCs). On recognition of its ligand, these receptors activate pAPCs, which may in turn influence naïve CD8+ T cell activation and affect their abilities to clear viral infection. However, how TLR ligands (TLR-L) can regulate CD8+ T cell responses have not been fully elucidated. This thesis will focus on examining how the presence of components from foreign pathogens, e.g. viral or bacterial infection, can contribute to shaping host immunity during concurrent viral infections. Since nitric oxide (NO), an innate effector immune molecule, was recently suggested to regulate proteasome activity; we sought to examine if NO can influence MHC-I antigen presentation during viral infections. The data in this section of the thesis provides evidence that combined TLR engagement can alter the presentation of certain CD8+ epitopes due to NO-induced inhibition in proteasome activity. Taken together, the data demonstrate that TLR ligation can influence the adaptive immune response due to induction of specific innate effector molecules such as NO. Next, the influence of combined TLR engagement on CD8+ T cell immunodominance hierarchies during viral infections was examined. In this section, we established that dual TLR2 and TLR3 stimulation alters immunodominance hierarchies of LCMV epitopes as a result of reduced uptake of cell-associated antigens and reduced cross-presentation of NP396 consequently suppressing NP396-specific CD8+ T cell responses. These findings are significant as they highlight a new role for TLR ligands in regulating anti-viral CD8+ T cell responses through impairing cross-presentation of cell-associated antigens depending on the type of TLR present in the environment during infections. Finally, we addressed TLR ligand induced type I interferon production and the signalling pathways that regulate them in two different mouse macrophage populations – those derived from the spleen or bone marrow. In this study, we observed that concomitant TLR2 stimulation blocked the induction of type I IFN induced by TLR4 in bone marrow-derived macrophages, but not spleen-derived macrophages in SOCS3-dependent manner. Taken together, the data presented in this thesis have defined new facets of how anti-viral responses are regulated by TLR activation, especially if multiple receptors are engaged simultaneously.

Regulation of the Gene Encoding Thrombin-Activable Fibrinolysis Inhibitor in Non-Hepatic Cells

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like pro-enzyme that, once activated, attenuates fibrinolysis. TAFIa also possesses anti-inflammatory properties. Although liver is the main source of plasma TAFI, platelet-derived TAFI has also been reported. An alternatively spliced TAFI variant resulted from the skipping of exon 6 and a 52-base deletion in exon 10 of CPB2 mRNA (∆6+10) was described to be brain specific. This TAFI variant is reputed to possess a secretase-like activity that cleaves β-amyloid precursor protein to form β-amyloid, a process involved in the onset of Alzheimer's disease. In this thesis, we report the identification of CPB2 mRNA and TAFI protein in various vascular and inflammatory cells. Specifically, we describe the expression of CPB2 mRNA in the megakaryocytic cell lines MEG-01 and Dami, the monocytic cell line THP-1, and peripheral blood mononuclear cells. TAFI protein was detected in differentiated Dami and THP-1 cells. We next describe the effect of external stimuli such as phorbol myristate acetate (PMA) on CPB2 expression in Dami and THP-1 cells. We found that PMA treatment increases both CPB2 mRNA abundance and promoter activity in Dami cells, and decreases both CPB2 mRNA abundance and promoter activity in THP-1 cells. Deletion analysis of the CPB2 promoter indicated cell-type specific regulation of CPB2 gene expression. Finally, we evaluated the expression of alternatively spliced CPB2 mRNA variants in hepatic and non hepatic cells. We found that exon 6 skipping variants are expressed in all cell types of interest. The variant previously reported to be brain specific was also found to be expressed in platelets. We found that the alternatively spliced TAFI variants accumulated inside the cells in a non-secretable, hypoglycosylated form and showed no carboxypeptidase activity. Taken together, this thesis provides further evidence supporting the hypothesis that platelet-derived TAFI is originated from CPB2 gene expression in megakaryocytes. Moreover, our data imply a potential for site-specific anti-inflammatory control provided by macrophage-derived TAFI. Alternative splicing of the CPB2 mRNA may give rise to variants with an intracellular role, perhaps as a peptidase chaperone, and may modulate the synthesis of secretable TAFI.

THE INFORMATION ERA THREATENS PRIVACY: A Comparative Study of Electronic Money’s Privacy Policies and Privacy Laws

This thesis consists of an analysis of electronic money (e-money), e-money’s privacy policies and relevant privacy laws. The value of information and the development of technology enhance the risk of privacy violations in the information era. Consumer privacy interests with respect to e-money are governed in part by the Personal Information Protection and Electronic Documents Act (PIPEDA) in Canada and by the European Union’s Data Protection Directive. The analysis is directed at whether the privacy policies of three kinds of e-money – Octopus Card, PayPal and MasterCard – comply with the spirit and letter of these laws. In light of technology change, the laws should be interpreted to apply broadly to protect privacy interests. Enhanced privacy protection may in fact lead to greater adoption of e-money by consumers.

Sequence and Structure Based Protein Folding Studies With Implications

As the expression of the genetic blueprint, proteins are at the heart of all biological systems. The ever increasing set of available protein structures has taught us that diversity is the hallmark of their architecture, a fundamental characteristic that enables them to perform the vast array of functionality upon which all of life depends. This diversity, however, is central to one of the most challenging problems in molecular biology: how does a folding polypeptide chain navigate its way through all of the myriad of possible conformations to find its own particular biologically active form? With few overarching structural principles to draw upon that can be applied to all protein architecture, the search for a solution to the protein folding problem has yet to produce an algorithm that can explain and duplicate this fundamental biological process. In this thesis, we take a two-pronged approach for investigating the protein folding process. Our initial statistical studies of the distributions of hydrophobic and hydrophilic residues within α-helices and β-sheets suggest (i) that hydrophobicity plays a critical role in helix and sheet formation; and (ii) that the nucleation of these motifs may result in largely unidirectional growth. Most tellingly, from an examination of the amino acids found in the smallest β-sheets, we do not find any evidence of a β-nucleating code in the primary protein sequence. Complementing these statistical analyses, we have analyzed the structural environments of several ever-widening aspects of protein topology. Our examination of the gaps between strands in the smallest β-sheets reveals a common organizational principle underlying β-formation involving strands separated by large sequential gaps: with very few exceptions, these large gaps fold into single, compact structural modules, bringing the β-strands that are otherwise far apart in the sequence close together in space. We conclude, therefore, that β-nucleation in the smallest sheets results from the co-location of two strands that are either local in sequence, or local in space following prior folding events. A second study of larger β-sheets both corroborates and extends these findings: virtually all large sequential gaps between pairs of β-strands organize themselves into an hierarchical arrangement, creating a bread-crumb model of go-and-come-back structural organization that ultimately juxtaposes two strands of a parental β-structure that are far apart in the sequence in close spatial proximity. In a final study, we have formalized this go-and-come-back notion into the concept of anti-parallel double-strandedness (DS), and measure this property across protein architecture in general. With over 90% of all residues in a large, non-redundant set of protein structures classified as DS, we conclude that DS is a unifying structural principle that underpins all globular proteins. We postulate, moreover, that this one simple principle, anti-parallel double-strandedness, unites protein structure, protein folding and protein evolution.

Flavian laws, the railway, the newspaper and Loring’s breads

It is abound the research on the formation, rise and failure of the financial and industrial network undertaken by the Loring-Heredia-Larios triangle, bourgeois families who introduced the Industrial Revolution in the south of Andalusia. On the contrary, there are almost nonexistent studies from the perspective of the mentality that sustained their business, social and ethical model in the algid decades of their action (1850-1860). In this paper we propose some hypotheses about the ideological structures of bourgeois group and point out some keys, clues and signs for a future reconstruction of this kind, which so far has not been incardinated that early and failed malaguenan industrial revolution in streams thinking of that time.

Accelerated apoptosis in SLE neutrophils cultured with anti-dsDNA antibody isolated from SLE patient serum: a pilot study.

Increased numbers of apoptotic neutrophils are found in SLE, related to disease activity and levels of anti-dsDNA antibody. The mechanism of increased apoptosis is not clear, but anti-dsDNA antibody has been shown to induce apoptosis in neutrophils from normal subjects and in certain cell lines. In this study, polyclonal anti-dsDNA antibody was isolated from the serum of a patient with active SLE, and was shown to substantially accelerate apoptosis in neutrophils from SLE patients as compared with neutrophils from healthy control or rheumatoid arthritis subjects.

Prevention of retinal capillary basement membrane thickening in diabetic dogs by a non-steroidal anti-inflammatory drug

AIMS/HYPOTHESIS: To investigate the effect of treatment with the non-steroidal anti-inflammatory drug Sulindac on the early vascular pathology of diabetic retinopathy in the dog, and it's effect on recognised biochemical indices of hyperglycaemia-related pathophysiology. METHODS: Experimental diabetes (streptozotocin/alloxan) was induced in 22 male beagle dogs and 12 of the animals were assigned at random to receive oral Sulindac (10 mg/kg daily). Age- and sex-matched control animals were maintained as non-diabetic controls. After 4 years, several morphological parameters were quantified in the retinal microvasculature of each animal group using an established stereological method. Also, the following diabetes-associated biochemical parameters were analysed: accumulation of advanced glycation end products (AGEs), red blood cell polyol levels and antioxidant status. RESULTS: Diabetes increased red blood cell sorbitol levels when compared to non-diabetic controls (p<or =0.05), however, there was no difference in sorbitol levels between the untreated and the treated diabetic animals. No significant differences were found in red blood cell myoinositol levels between the three groups of animals. Pentosidine and other AGEs were increased two- to three-fold in the diabetic animals (p<or =0.001) although treatment with Sulindac did not affect their accumulation in diabetic skin collagen or alter diabetes-induced rises in plasma malondialdehyde. Retinal capillary basement membrane volume was significantly increased in the untreated diabetic dogs compared to non-diabetic controls or Sulindac-treated diabetic animals (p<or =0.0001). CONCLUSION/INTERPRETATION: This study has confirmed the beneficial effect of a non-steroidal anti-inflammatory drug on the early vascular pathology of diabetic retinopathy. However the treatment benefit was not dependent on inhibition of polyol pathway activity, advanced glycation, or oxidative stress.

Characterisation and evaluation of novel surfactant bacterial anti-adherent coatings for endotracheal tubes designed for the prevention of ventilator-associated pneumonia.

It is accepted that ventilator-associated pneumonia is a frequent cause of morbidity and mortality in intensive care patients. This study describes the physicochemical properties of novel surfactant coatings of the endotracheal tube and the resistance to microbial adherence of surfactant coated endotracheal tube polyvinylchloride (PVC). Organic solutions of surfactants containing a range of ratios of cholesterol and lecithin (0:100, 25:75, 50:50, 75:25, dissolved in dichloromethane) were prepared and coated onto endotracheal tube PVC using a multiple dip-coating process. Using modulated temperature differential scanning calorimetry it was confirmed that the binary surfactant systems existed as physical mixtures. The surface properties of both surfactant-coated and uncoated PVC, following treatment with either pooled human saliva or phosphate-buffered saline (PBS), were characterised using dynamic contact angle analysis. Following treatment with saliva, the contact angles of PVC decreased; however, those of the coated biomaterials were unaffected, indicating different rates and extents of macromolecular adsorption from saliva onto the coated and uncoated PVC. The advancing and receding contact angles of the surfactant-coated PVC were unaffected by sonication, thereby providing evidence of the durability of the coatings. The cell surface hydrophobicity and zeta potentials of isolates of Staphylococcus aureus and Pseudomonas aeruginosa, following treatment with either saliva or PBS, and their adherence to uncoated and surfactant-coated PVC (that had been pre-treated with saliva) were examined. Adherence of S. aureus and Ps. aeruginosa to surfactant-coated PVC at each successive time period (0.5, 1, 2, 4, 8 h) was significantly lower than to uncoated PVC, the extent of the reduction frequently exceeding 90%. Interestingly, the microbial anti-adherent properties of the coatings were dependent on the lecithin content. Based on the impressive microbial anti-adherence properties and durability of the surfactant coating on PVC following dip coatings, it is proposed that these systems may usefully reduce the incidence of ventilator-associated pneumonia when employed as luminal coatings of the endotracheal tube.