Biological membranes form a protective barrier around cells and cellular compartments. A broad range of biochemical processes occur in or at membranes demonstrating that they are not only of structural but also of functional importance. One important class of membrane proteins are membrane-associated glycosyltransferases. WaaG is a representative of this class of proteins; its function is to catalyze one step in the synthesis of lipopolysaccharides, which are outer membrane lipids found in Gram-negative bacteria. To study protein-membrane complexes by biophysical methods, one must employ membrane mimetics, i.e. simplifications of natural membranes. One type of membrane mimetic often employed in solution-state NMR is small isotropic bicelles, obloid aggregates formed from a lipid bilayer that is dissolved in aqueous solvent by detergent molecules that make up the rim of the bicelle. In this thesis, fast dynamics of lipid atoms in bicelles containing lipid mixtures that faithfully mimic plant and bacterial membranes were investigated by NMR relaxation. Lipids were observed to undergo a broad range of motions; while the glycerol backbone was found to be rigid, dynamics in the acyl chains were much more rapid and unrestricted. Furthermore, by employing paramagnetic relaxation enhancements an ‘atomic ruler’ was developed that allows for measurement of the immersion depths of lipid carbon atoms. WaaG is a membrane-associated protein that adopts a GT-B fold. For proteins of this type, it has been speculated that the N-terminal domain anchors tightly to the membrane via electrostatic interactions, while the anchoring of the C-terminal domain is weaker. Here, this model was tested for WaaG. It was found by a set of circular dichroism, fluorescence, and NMR techniques that an anchoring segment located in the N-terminal domain termed MIR-WaaG binds electrostatically to membranes, and the structure and localization of isolated MIR-WaaG inside micelles was determined. Full-length WaaG was also found to bind membranes electrostatically. It senses the surface charge density of the membrane whilst not discriminating between anionic lipid species. Motion of the C-terminal domain could not be observed under the experimental conditions used here. Lastly, the affinity of WaaG to membranes is lower than expected, indicating that WaaG should not be classified as a monotopic membrane protein but rather as a peripheral one. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Manuscript.</p>

Carbohydrates are ubiquitous components in nature involved in a range of tasks. They cover every cell and contribute both structural stability as well as identity. Lipopolysaccharides are the outermost exposed part of the bacterial cell wall and the primary target for host-pathogen recognition. Understanding the structure and biosynthesis of these polysaccharides is crucial to combat disease and develop new medicine. Structural determinations can be carried out using NMR spectroscopy, a powerful tool giving information on an atomistic scale. This thesis is focused on method development to study polysaccharide structures as well as application on bacterial lipopolysaccharides. The focus has been to incorporate a bioinformatics approach prior to analysis by NMR spectroscopy, and then computer assisted methods to aid in the subsequent analysis of the spectra. The third chapter deals with the recent developments of ECODAB, a tool that can help predict structural fragments in Escherichia coli O-antigens. It was migrated to a relational database and the aforementioned predictions can now be made automatically by ECODAB. The fourth chapter gives insight into the program CASPER, a computer program that helps with structure determination of oligo- and polysaccharides. An approach to determine substituent positions in polysaccharides was investigated. The underlying database was also expanded and the improved capabilities were demonstrated by determining O-antigenic structures that could not previously be solved. The fifth chapter is an application to O‑antigen structures of E. coli strains. This is done by a combination of NMR spectroscopy and bioinformatics to predict components as well as linkages prior to spectra analysis. In the first case, a full structure elucidation was performed on E. coli serogroup O63, and in the second case a demonstration of the bioinformatics approach is done to E. coli serogroup O93. In the sixth chapter, a new version of the CarbBuilder software is presented. This includes a more robust building algorithm that helps build sterically crowded polysaccharide structures, as well as a general expansion of possible components. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 5: Manuscript.</p>

6 Abstract Lipopolysaccharides are composed of covalently bound saccharides. They are a characteristic component of the cell wall of gram-negative bacteria. They are the cause of severe sepsis in humans and complications in human medicine. Lipopolysaccharides are a constant part of the infections of gram-negative bacteria. We expect an evolutionarily conserved non-specific immune response and protection. The question is whether there is an immune response in the model organism Caenorhabditis elegans. If so, what mechanism is controlled and regulated. We submitted lipopolysaccharides from the bacteria Pseudomona aeruginosa with the bacteria Escherichia coli OP50 and observed the influence of lipopolysaccharides on the expression of selected genes. We examined metabolism and development. We have shown the influence of lipopolysaccharides on gene expression of C-type lectine clec-60 a clec-71, nextna lys-5, hsp-60 a F44G.3.2.1 genes. We incubated Caenorhabditis elegans on some components of lipopolysaccharide. We found regulation of these selected genes with hydrophobic components of lipopolysacharide, lipid A. We did not observe regulation with saccharide components of lipopolysaccharide, glucose and galatose. The metabolism of lipids had changed. We demonstrated a reduction of neutral lipids and changes in...

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Puberty is a critical period for sexual maturation during which the sex-specific reorganization and remodelling of the pubertal brain facilitate sex biases in stress sensitivity. Pubertal (i.e., six-week-old) CD-1 mice treated with the bacterial endotoxin lipopolysaccharide (LPS; 1.5 mg/kg body weight, ip) show several sex-specific changes to the neuroendocrine and behavioural systems of several reproductive and non-reproductive functions. One promising explanation for the elusive mechanisms driving the sex-specific outcomes of pubertal immune challenge may lie in the cascade of neuroimmune events induced by this systemic immune stressor. This doctoral thesis tested the hypothesis that sex-specific responses of the pubertal neuroimmune network contribute to sex differences in the enduring outcomes of pubertal immune challenge on hippocampus-dependent cognitive processes. Male and female CD-1 mice are equally vulnerable to enduring impairments in spatial memory following pubertal LPS exposure. Across brain regions for cognition and stress regulation, pubertal LPS treatment alters baseline sex differences in microglial expression and morphology in a sex-dependent manner. The temporary female-specific increase in whole-brain blood-brain barrier permeability during LPS-induced sickness may have facilitated the apparent female bias in LPS-induced changes to pubertal microglia. In the context of sex- and region-specific residual effects of pubertal LPS-induced sickness on microglial expression and morphology, pubertal LPS treatment may accelerate certain neurodevelopmental processes in males but not females. The innate sex differences in the pubertal neuroimmune network highlighted by these studies underscore how a systemic immune challenge precipitates sex biases in immune-mediated disorders of brain and behaviour during adulthood.

Indiana University-Purdue University Indianapolis (IUPUI) / Embryonic stem cells (ESCs) are unique in that they have potential to give rise to every cell type of the body. Little is known about stimuli that promote mouse (m)ESC differentiation and proliferation. Therefore the purpose of this study was to determine the role of Toll Like Receptor (TLR) ligands in mESCs proliferation, survival, and differentiation in the presence of Leukemia Inhibitory Factor (LIF). We hypothesized that TLRs are expressed and functional, and when activated by their ligand will induce survival, proliferation, and prevent differentiation. In this study, mESC line E14 was used to determine the expression of TLRs at the mRNA level and three mESC lines, R1, CGR8, and E14, were used to determine cell surface protein levels. We found expression of TLRs 1, 2, 3, 5, and 6 at the mRNA level, but no expression of TLRs 4, 7, 8, and 9 in the E14 mESC line. We confirmed the presence of TLR-2 but not of TLR-4, protein on the cell surface using flow cytometric analysis for all three cell lines. We focused our studies mainly on TLR-2 using the E14 cell line. Pam3Cys, is a synthetic triacyl lipoprotein and a TLR-2 ligand, which induced a significant increase in mESC proliferation on Days 3, 4, and 5 and enhanced survival of mESC in a dose dependent manner in the context of delayed addition of serum. All the latter experiments were performed in triplicate and student T-test was performed to establish significant differences. Next, we demonstrated functionality of TLR-2 via the MyD88/IKK pathway, where MyD88 was expressed and IKKα/β phosphorylation was enhanced. This was associated with increased NF-κB nuclear translocation upon activation by Pam3Cys. Finally, we showed that there were no changes in expression of mESCs markers Oct-4, KLF-4, Sox-2, and SSEA-1, thus illustrating that the mESCs may have remained in a pluripotent state after activation with the TLR-2 ligand in the presence of LIF. These results demonstrate that mESCs can respond to microbial products, such as Pam3Cys, and can induce proliferation and survival of the mESCs. This finding expands the role of TLRs and has some implications in understanding embryonic stem cell biology.

Tryptophanis anaminoacidwhichhasmanyfunctionsinthebody. Besidesitsparticipationin theproductionofproteins,itactsasasubstrateforthekynurenineandmethoxyindolemetabolic pathways. The kynurenine pathway ends with the production of nikotinamid e adenin dinukleotide ( NAD + ) ,whichisneededfortheproductionofcellularenergy. Thus,withincreased energy demand during immune system activation, the activity of the kynurenine pathway is increased. Dueto increasedactivity,itproduces more immunoactiveandneuroactivemetab olites suchaskynurenicacidandquinolinicacid.Thesemetabolitesareinvolvedinmanyprocessesin the body and affect the pathology of many diseases. Studies show that regulation of these metabolites could be a key innovation in the treatment of cance r, cardiovascular or neurodegenerative diseases. The methoxyindole pathway is another important tryptophan processingpathway.Itsbest - knownmetabolitesareserotonin,whichactsasaneurotransmitter, andmelatonin,ahormonewithimmunomodulatoryeffect sregulatedby thecircadianclock . This workdeals with thecircadian rhythmicityofenzymeexpressionofthesetwo metabolic pathways.Italsodescribestheeffectofsystemicadministrationoflipopolysaccharideendotoxin ontheexpressionofgenesoft heseenzymes.OurexperimentsusedWistarratsat30daysofage. The lipopolysaccharide was administered...

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The liver regulates inflammation during sepsis, and most liver functions are carried out by hepatocytes. Bacterial lipids, including lipopolysaccharide (LPS) and lipoteichoic acid (LTA), can be cleared by hepatocytes, but the underlying mechanisms are uncertain. Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates uptake of LPS by hepatocytes, but it is unknown whether LTA uptake is similarly regulated. Therefore, our objectives were to characterize the PCSK9-regulated pathway of bacterial lipid uptake by hepatocytes by identifying whether low-density lipoprotein (LDL) receptor (LDLR) and LDLR-related protein 1 (LRP1) are the target receptors, and by determining which lipoproteins are involved. To study this pathway, we assessed the uptake of fluorescently-labeled LPS or LTA by human HepG2 hepatocytes using flow cytometry. We pre-treated HepG2 cells with PCSK9, alone or in combination with anti-LDLR or anti-LRP1 antibodies, in order to identify the PCSK9-regulated receptors that are involved, and utilized media containing normal serum or lipoprotein-deficient serum to investigate the lipoprotein- dependence of this pathway. We also determined the roles of LDL and HDL in bacterial lipid uptake through a series of add-back experiments to lipoprotein-deficient serum, and blocked LDLR to confirm that LDLR mediates LDL-dependent uptake. The HepG2 cell response to variable degrees of bacterial lipid uptake was also assessed in a subset of experiments by measuring several cytokines and extracellular alanine aminotransferase (ALT) activity in the cell culture supernatant. We found that PCSK9 regulates LDLR-mediated uptake of both LPS and LTA through an LDL-dependent mechanism, while LRP1 is not involved. Increased bacterial lipid uptake did not result in any hepatocellular injury or cytokine production, as measured by ALT activity and interleukin (IL)-6, IL-8, IL-10, and IL-17 concentrations. In conclusion, we completed our objective of characterizing the PCSK9-regulated pathway of bacterial lipid uptake, and provide supporting evidence for targeting PCSK9 as a novel therapeutic avenue in sepsis. / Thesis / Master of Science (MSc) / Bacterial compounds stimulate inflammation that can be overwhelming during sepsis. Understanding the processes behind uptake and clearance of these compounds may lead to better sepsis treatments. Therefore, our goal was to understand how uptake of two bacterial compounds, lipopolysaccharide and lipoteichoic acid, occurs by liver cells called hepatocytes. Hepatocytes are naturally equipped to clear foreign compounds, so understanding their role in clearing bacterial compounds is important. Another goal was to identify the role of the protein PCSK9 in this uptake process, as treatments targeting PCSK9 could be applied to sepsis once we understand its role in this disease. Our research demonstrates the negative role of PCSK9 in regulating uptake of lipopolysaccharide and lipoteichoic acid through a lipoprotein receptor called LDLR, and identifies the role of lipoproteins in this process. These findings further our understanding of the hepatocyte response to bacterial compounds in relation to sepsis, and identify PCSK9 as a potential target for new sepsis therapies.